Novel peptides and scaffolds for use in immunotherapy against head and neck squamous cell carcinoma and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/750,908, filed 23 Jan. 2020, which is a continuation of U.S.application Ser. No. 16/422,335, filed 24 May 2019, now U.S. Pat. No.10,596,196, issued 24 Mar. 2020, which is a Continuation of U.S.application Ser. No. 15/686,679, filed 25 Aug. 2017, now U.S. Pat. No.10,376,542, issued 13 Aug. 2019, which claims the benefit of U.S.Provisional Application Ser. No. 62/379,864, filed 26 Aug. 2016, andGerman Application No. 102016115974.3, filed 26 Aug. 2016, the contentof each of these applications is herein incorporated by reference intheir entirety.

This application also is related to PCT/EP2017/071347 filed 24 Aug.2017, the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-075009_ST25.txt” createdon 2 Apr. 2020, and 25,203 bytes in size) is submitted concurrently withthe instant application, and the entire contents of the Sequence Listingare incorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

Head and neck squamous cell carcinomas (HNSCC) are heterogeneous tumorswith differences in epidemiology, etiology and treatment (Economopoulouet al., 2016). These tumors are categorized by the area in which theybegin. They include cancers of the oral cavity (lips, front two-thirdsof the tongue, gums, lining inside the cheeks and lips, floor of themouth, hard palate), the pharynx (nasopharynx, oropharynx including softpalate, base of the tongue, tonsils, hypopharynx), larynx, paranasalsinuses and nasal cavity and salivary glands (National Cancer Institute,2015).

HNSCC is the sixth most common malignancy in the world and accounts forabout 6% of all cancer cases diagnosed worldwide (Economopoulou et al.,2016). HNSCC is characterized by a wide geographical variation in theincidence and anatomic distribution (Vigneswaran and Williams, 2014).High risk countries are located in South and Southeast Asia (i.e. India,Sri Lanka, Bangladesh, Pakistan). In these regions, squamous cellcarcinoma of the oral cavity (OSCC) is the most common cancer in men andthe third most common cancer in women (Vigneswaran and Williams, 2014).In Europe, high incidence rates of OSCC are found in regions of France,Hungary, Slovakia and Slovenia. In the United States, HNSCC is theeighths most common cancer among men.

Major risk factors for HNSCC are alcohol and tobacco use. Other HNSCCrisk factors include consumption of maté, but also of preserved orsalted foods, use of betel quid, occupational exposure to wood dust,asbestos and synthetic fibers, radiation exposure, infection withcancer-causing types of human papillomavirus (HPV) or Epstein-Barr virus(EBV) and ancestry (particularly Chinese ancestry in nasopharyngeal SCC)(National Cancer Institute, 2015).

While OSCC and laryngeal SCC have decreased in developed countries, theincidence of oropharyngeal SCC has increased. This is attributed to achange in the biologic driver of SCC (HPV-related SCC instead ofsmoking-related SCC). HPV-related oropharyngeal cancers have increasedby 225% from 1988 to 2004 (National Cancer Institute, 2015).HPV-positive HNSCC may represent a distinct disease entity. Those tumorsare associated with significantly improved survivals.

Rates of incidence depend on gender: male to female ratio ranges from2:1 to 4:1 (2014 Review of cancer Medicines on the WHO list of essentialmedicines). The five-year overall survival rate of patients with HNSCCis 40-50% (World Health Organization, 2014). While early cancers (T1,T2) have cure rates of 70%-95% (Nat Cancer Inst), the majority ofpatients with HNSCC present with locally advanced disease (Bauml et al.,2016). Treatment for early HNSCC involves single-modality therapy witheither surgery or radiation (World Health Organization, 2014). Advancedcancers are treated by a combination of chemotherapy with surgery and/orradiation therapy.

Chemotherapy includes mostly cisplatin or drug combinations that containcisplatin like docetaxel, cisplatin, fluorouracil (5-FU) or cisplatin,epirubicin, bleomycin or cisplatin, 5-FU. Isotretinoin (13-cis-retinoicacid) is used in oral cavity SCC and laryngeal SCC, daily for 1 year toreduce the incidence of second tumors (National Cancer Institute, 2015).

HNSCC is considered an immunosuppressive disease, characterized by thedysregulation of immunocompetent cells and impaired cytokine secretion(Economopoulou et al., 2016). Immunotherapeutic strategies differbetween HPV-negative and HPV-positive tumors.

In HPV-positive tumors, the viral oncoproteins E6 and E7 represent goodtargets, as they are continuously expressed by tumor cells and areessential to maintain the transformation status of HPV-positive cancercells. Several vaccination therapies are currently under investigationin HPV-positive HNSCC, including DNA vaccines, peptide vaccines andvaccines involving dendritic cells (DCs). Additionally, an ongoing phaseII clinical trial investigates the efficacy of lymphodepletion followedby autologous infusion of TILs in patients with HPV-positive tumors(Economopoulou et al., 2016).

In HPV-negative tumors, several immunotherapeutic strategies arecurrently used and under investigation. The chimeric IgG1 anti-EGFRmonoclonal antibody cetuximab has been approved by the FDA incombination with chemotherapy as standard first line treatment forrecurring/metastatic HNSCC. Other anti-EGFR monoclonal antibodies,including panitumumab, nimotuzumab and zalutumumab, are evaluated inHNSCC.

Several immune checkpoint inhibitors are investigated in clinical trialsfor their use in HNSCC. They include the following antibodies:Ipilimumab (anti-CTLA-4), tremelimumab (anti-CTLA-4), pembrolizumab(anti-PD-1), nivolumab (anti-PD-1), durvalumab (anti-PD-1), anti-KIR,urelumab (anti-CD137), and anti-LAG-3.

Two clinical studies with HNSCC patients evaluated the use of DCs loadedwith p53 peptides or apoptotic tumor cells. The immunological responseswere satisfactory and side effects were acceptable.

Several studies have been conducted using adoptive T cell therapy (ACT).T cells were induced against either irradiated autologous tumor cells orEBV. Results in disease control and overall survival were promising(Economopoulou et al., 2016).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and head and neck squamous cell carcinomain particular. There is also a need to identify factors representingbiomarkers for cancer in general and head and neck squamous cellcarcinoma in particular, leading to better diagnosis of cancer,assessment of prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.

c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.

e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of immense importance for the developmentof pharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006). Elongated (longer) peptides of the invention can act as MHC classII active epitopes. T-helper cells, activated by MHC class II epitopes,play an important role in orchestrating the effector function of CTLs inanti-tumor immunity. T-helper cell epitopes that trigger a T-helper cellresponse of the TH1 type support effector functions of CD8-positivekiller T cells, which include cytotoxic functions directed against tumorcells displaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-Class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way, each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated und thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 91 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 91, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 91 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 91,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 have been disclosed beforein large listings as results of high-throughput screenings with higherror rates or calculated using algorithms, but have not been associatedwith cancer at all before. The peptides in Table 3 are additionalpeptides that may be useful in combination with the other peptides ofthe invention. The peptides in Table 4A and B are furthermore useful inthe diagnosis and/or treatment of various other malignancies thatinvolve an over-expression or over-presentation of the respectiveunderlying polypeptide.

TABLE 1 Peptides according to the present invention. SEQ ID No. SequenceGene ID(s) Official Gene Symbol(s) 1 GLAGGFGGPGFPV 286887, 3853, 3854KRT6C, KRT6A, KRT6B 2 PVCPPGGIQEV 286887, 3848, 3852, KRT6C, KRT1, KRT5,3853, 3854, 9119 KRT6A, KRT6B, KRT75 3 SLYGLGGSKRISI 286887, 3853, 3854KRT6C, KRT6A, KRT6B 4 ILDINDNPPV 100653137, 1830, DSG3, CDH23 64072 5VCPPGGIQEV 286887, 3848, 3852, KRT6C, KRT1, KRT5, 3853, 3854, 9119KRT6A, KRT6B, KRT75 6 ALYDAELSQM 286887, 3853, 3854 KRT6C, KRT6A, KRT6B7 ALEEANADLEV 3860, 3861, 3866, KRT13, KRT14, KRT15,3868, 400578, 644945, KRT16, KRT16P2, 729252 KRT16P3, KRT16P1 8AQLNIGNVLPV 6132 RPL8 9 STASAITPSV 3852 KRT5 10 TLWPATPPKA 647024C6orf132 11 VLFSSPPVI 5621 PRNP 12 TLTDEINFL 286887, 3853, 3854KRT6C, KRT6A, KRT6B 13 SLVSYLDKV unknown gene 14 RIMEGIPTV 242 ALOX12B15 SMLNNIINL 5317 PKP1 16 ALKDSVQRA 10765 KDM5B 17 SIWPALTQV 100381270ZBED6 18 YLYPDLSRL 6538 SLC6A11 19 ALAKLLPLL 5655 KLK10 20 YLINEIDRIRA667 DST 21 FLHEPFSSV 122665, 84659 RNASE8, RNASE7 22 KLPEPCPSTV 6707SPRR3 23 SLPESGLLSV 2178 FANCE 24 LLIAINPQV 9635 CLCA2 25 SLCPPGGIQEV196374 KRT78 26 TLVDENQSWYL 341208 HEPHL1 27 YLAEPQWAV 2196 FAT2 28AVDPVSGSLYV 57451 TENM2 29 RLLPDLDEV 121551 BTBD11 30 TLASLGYAVV 91039DPP9 31 HLATVKLLV 54101 RIPK4 32 IQDAEGAIHEV 165904 XIRP1 33 AIYEGVGWNV57115 PGLYRP4 34 ALDTFSVQV 171177 RHOV 35 ALVGDVILTV 2196 FAT2 36GLWSSIFSL 123745 PLA2G4E 37 ILLEDVFQL 285973 ATG9B 38 KLLPGVQYV 390928PAPL 39 LLPEDDTRDNV 1001 CDH3 40 LLTPLNLQI 286887, 3852, 3853,KRT6C, KRT5, KRT6A, 3854 KRT6B 41 RLNGEGVGQVNISV 3853, 3854 KRT6A, KRT6B42 ALYTSGHLL 5653 KLK6 43 AVLGGKLYV 9903 KLHL21 44 GLGDDSFPI 2125 EVPL45 GLIEWLENTV 5591 PRKDC 46 GLISSIEAQL 3860 KRT13 47 QLLEGELETL 5493 PPL48 YLLDYPNNL 26057 ANKRD17 49 YLWEAHTNI 729830 FAM160A1 50 ALSNVVHKV5268 SERPINB5 51 FLIPSIIFA 150696 PROM2 52 LLFTGLVSGV 284434 NWD1 53RLVEVGGDVQL 3963, 653499 LGALS7, LGALS7B 54 RLSGEGVGPV 3852 KRT5 55VLNVGVAEV 285848 PNPLA1 56 FLQLETEQV 64426 SUDS3 57 AILGFALSEA516, 517, 518 ATP5G1, ATP5G2, ATP5G3 58 SLSDIQPCL 3691 ITGB4 59YLQNEVFGL 1832 DSP 60 SLGNFKDDLL 23650 TRIM29 61 FVAGYIAGV 5250 SLC25A362 ILSSACYTV 5317 PKP1 63 ALMDEINFMKM 3852 KRT5 64 KILEJLFVJLunknown gene 65 ALWGFFPVLL 56851 EMC7 66 TLLSEIAEL 84629 TNRC18 67AQLNLIWQL 80381 CD276 68 KILEMDDPRA 6512 SLC1A7 69 YVMESMTYL 28976 ACAD970 FLFPAFLTA 2150 F2RL1 71 SLFPYVVLI 55117 SLC6A15 72 SLDGNPLAV 25987TSKU 73 YIDPYKLLPL 54433 GAR1 74 SLTSFLISL 101060198, 7851 MALL 75ALASAPTSV 80004 ESRP2 76 ILFDEVLTFA 83666 PARP9 77 SLRAFLMPI 79901CYBRD1 78 VLYGDVEEL 10970 CKAP4 79 GLHQDFPSVVL 51056 LAP3 80 GLYGIKDDVFL3939 LDHA J = phospho-serine

TABLE 2Additional peptides according to the present invention with no prior known cancerassociation. SEQ ID No. Sequence Gene ID(s) Official Gene Symbol(s) 81VLAENPDIFAV 6541 SLC7A1 82 VLDINDNPPV 120114, 1828, 2195FAT3, DSG1, FAT1 83 QLLQYVYNL 4173 MCM4 84 ALMAGCIQEA 1026 CDKN1A 85QLIEKITQV 114827 FHAD1 86 SLQERQVFL 9333 TGM5 87 ALPEPSPAA 5339 PLEC 88LMAPAPSTV 7071 KLF10 89 VLDEGLTSV 25909, 285116 AHCTF1, AHCTF1P1 90TLNDGVVVQV 10146 G3BP1 91 MLFENMGAYTV 4953 ODC1

TABLE 3 Peptides useful for e.g. personalized cancer therapies. SEQID No. Sequence Gene ID(s) Official Gene Symbol(s) 92 ILLDVKTRL3728, 3861, 3868, 3872 JUP, KRT14, KRT16, KRT17 93 ALSNVIHKV 5268SERPINB5 94 SIFEGLLSGV 2709 GJB5 95 SLDENSDQQV 6273 S100A2 96FQLDPSSGVLVTV 2196 FAT2 97 LILESIPVV 5597 MAPK6 98 SLYKGLLSV 25788RAD54B 99 TASAITPSV 3852 KRT5 100 VLVSDGVHSV 1952 CELSR2 101 GLLPSAESIKL132989 C4orf36 102 TLAELQPPVQL 157922 CAMSAP1 103 VLAEGGEGV 10630 PDPN104 SLSPVILGV 26525 IL36RN 105 STYGGGLSV 3861, 3868 KRT14, KRT16 106VLVDQSWVL 5655 KLK10 107 YLEEDVYQL 23255 SOGA2 108 SLYNLGGSKRISI 3852KRT5 109 KIQEILTQV 10643 IGF2BP3 110 LLPPPPPPA 9509 ADAMTS2 111SLAPGDVVRQV 79729 SH3D21 112 ALLDGGSEAYWRV 84985 FAM83A 113 NLMASQPQL5317 PKP1 114 VLVPYEPPQV 8626 TP63 115 VTAAYMDTVSL 7498 XDH 116SLWPSPEQL 90480 GADD45GIP1 117 GLAFSLYQA 871 SERPINH1 118 TLLQEQGTKTV286887, 3852, 3853, KRT6C, KRT5, KRT6A, 3854 KRT6B 119 GLLDPSVFHV 79050NOC4L 120 YLVAKLVEV 10277 UBE4B 121 SLYGYLRGA 9790 BMS1 122 ILDEAGVKYFL113828 FAM83F 123 LLSGDLIFL 2709 GJB5 124 YMLDIFHEV 3038 HAS3 125ALNPEIVSV 5277 PIGA 126 ILVDWLVEV 85417, 890, 8900 CCNB3, CCNA2, CCNA1127 SLFGKKYIL 2274 FHL2 128 TLHRETFYL 9134 CCNE2 129 SLSGEIILHSV 121441NEDD1 130 TLDGAAVNQV 3918 LAMC2 131 LQLDKEFQL 24140 FTSJ1 132 TLYPGRFDYV338322 NLRP10 133 LLLPLQILL 5650 KLK7 134 ILIGETIKI 5742, 5743PTGS1, PTGS2 135 GLFSQHFNL 1789 DNMT3B 136 SLMEPPAVLLL 8900 CCNA1 137GLAPFLLNAV 101060689, 154761, FAM115C 285966 138 ALLTGIISKA 23165 NUP205139 QLGPVPVTI 285966 FAM115C 140 YLFENISQL 57115 PGLYRP4 141 FLNPDEVHAI81610 FAM83D 142 SLVSEQLEPA 11187 PKP3 143 YVYQNNIYL 2191 FAP 144KISTITPQI 996 CDC27 145 LLYGKYVSV 84065 TMEM222 146 GLLEELVTV 642475MROH6 147 ILMDPSPEYA 1786 DNMT1 148 LLFDAPDLRL 55561 CDC42BPG 149VLLNINGIDL 222484 LNX2 150 ILAEEPIYIRV 55655 NLRP2 151 QLCDLNAEL 3833KIFC1 152 SLWQDIPDV 128272 ARHGEF19 153 VLFLGKLLV 204962 SLC44A5 154KMWEELPEVV 622 BDH1 155 GLLDNPELRV 26263 FBXO22 156 ALINDILGELVKL 85463ZC3H12C

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, acute myelogenousleukemia, breast cancer, bile duct cancer, brain cancer, chroniclymphocytic leukemia, colorectal carcinoma, esophageal cancer,gallbladder cancer, gastric cancer, hepatocellular cancer, melanoma,non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, renal cell cancer, small cell lungcancer, urinary bladder cancer and uterine cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 91. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 31 (see Table 1), and their uses in theimmunotherapy of head and neck squamous cell carcinoma, acutemyelogenous leukemia, breast cancer, bile duct cancer, brain cancer,chronic lymphocytic leukemia, colorectal carcinoma, esophageal cancer,gallbladder cancer, gastric cancer, hepatocellular cancer, melanoma,non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, renal cell cancer, small cell lungcancer, urinary bladder cancer, uterine cancer, and preferably head andneck squamous cell carcinoma.

As shown in the following Table 4, many of the peptides according to thepresent invention are also found on other tumor types and can, thus,also be used in the immunotherapy of other indications. Also refer toFIGS. 1A-1Q and Example 1.

TABLE 4APeptides according to the present invention and their specific uses in otherproliferative diseases, especially in other cancerous diseases. The table showsfor selected peptides on which additional tumor types they were found and eitherover-presented on more than 5% of the measured tumor samples, or presented onmore than 5% of the measured tumor samples with a ratio of geometric means tumorvs normal tissues being larger than 3. Over-presentation is defined as higherpresentation on the tumor sample as compared to the normal sample with highestpresentation. Normal tissues against which over-presentation was tested were:adipose tissue, adrenal gland, bile duct, blood cells, blood vessel, bone marrow,brain, esophagus, eye, gallbladder, heart, kidney, large intestine, liver, lung,lymph node, nerve, pancreas, parathyroid gland, peritoneum, pituitary, pleura,salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, thymus,thyroid gland, trachea, ureter, urinary bladder. SEQ ID No SequenceOther relevant organs/diseases  1 GLAGGFGGPGFPVGallbladder Cancer, Bile Duct Cancer  2 PVCPPGGIQEV NSCLC, SCLC  3SLYGLGGSKRISI Esophageal Cancer, Urinary bladder cancer  4 ILDINDNPPVBRCA, Esophageal Cancer, Urinary bladder cancer  7 ALEEANADLEVEsophageal Cancer, Urinary bladder cancer  8 AQLNIGNVLPVMelanoma, Esophageal Cancer  9 STASAITPSV Melanoma 10 TLWPATPPKAGallbladder Cancer, Bile Duct Cancer 11 VLFSSPPVI Melanoma 15 SMLNNIINLUrinary bladder cancer 16 ALKDSVQRAAML, BRCA, Melanoma, Esophageal Cancer, Urinary bladder cancer,Uterine Cancer 17 SIWPALTQVSCLC, AML, BRCA, Melanoma, Uterine Cancer, Gallbladder Cancer,Bile Duct Cancer 18 YLYPDLSRL Brain Cancer 19 ALAKLLPLLEsophageal Cancer, Urinary bladder cancer, Uterine Cancer 20 YLINEIDRIRANSCLC, Urinary bladder cancer 21 FLHEPFSSV Urinary bladder cancer 23SLPESGLLSV Esophageal Cancer 24 LLIAINPQV AML, Urinary bladder cancer 27YLAEPQWAV Esophageal Cancer, Urinary bladder cancer 29 RLLPDLDEV AML 31HLATVKLLV Urinary bladder cancer, Uterine Cancer 32 IQDAEGAIHEVNHL, Melanoma, Esophageal Cancer, Urinary bladder cancer 37 ILLEDVFQLGallbladder Cancer, Bile Duct Cancer 39 LLPEDDTRDNVBRCA, Esophageal Cancer, Urinary bladder cancer 41 RLNGEGVGQVNISVEsophageal Cancer 42 ALYTSGHLLEsophageal Cancer, Gallbladder Cancer, Bile Duct Cancer 43 AVLGGKLYVCLL, NHL, AML, Melanoma, Uterine Cancer 45 GLIEWLENTVSCLC, OC, Urinary bladder cancer 46 GLISSIEAQLEsophageal Cancer, Urinary bladder cancer, Uterine Cancer 47 QLLEGELETLUrinary bladder cancer, Uterine Cancer 48 YLLDYPNNLNHL, BRCA, Melanoma, Esophageal Cancer, Urinary bladder cancer,Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer 49 YLWEAHTNIEsophageal Cancer, Uterine Cancer 50 ALSNVVHKVGC, BRCA, Esophageal Cancer, Urinary bladder cancer 51 FLIPSIIFAUrinary bladder cancer, Uterine Cancer 52 LLFTGLVSGV Esophageal Cancer53 RLVEVGGDVQL Esophageal Cancer, Urinary bladder cancer 54 RLSGEGVGPVEsophageal Cancer 56 FLQLETEQVBRCA, Melanoma, Urinary bladder cancer, Uterine Cancer 57 AILGFALSEAAML, BRCA, Melanoma, Uterine Cancer, Gallbladder Cancer,Bile Duct Cancer 58 SLSDIQPCLBrain Cancer, BRCA, Esophageal Cancer, Urinary bladder cancer,Uterine Cancer 59 YLQNEVFGL SCLC, NHL 60 SLGNFKDDLLNHL, Esophageal Cancer, Urinary bladder cancer 61 FVAGYIAGVNSCLC, SCLC, RCC, Brain Cancer, GC, CLL, NHL, EsophagealCancer, Gallbladder Cancer, Bile Duct Cancer 62 ILSSACYTVRCC, BRCA, Esophageal Cancer 63 ALMDEINFMKM NSCLC, SCLC, NHL 64KILEJLFVJL NSCLC, Urinary bladder cancer, Gallbladder Cancer, Bile DuctCancer 65 ALWGFFPVLL CLL, Melanoma 66 TLLSEIAELCLL, NHL, AML, Melanoma, OC, Uterine Cancer 67 AQLNLIWQLSCLC, Melanoma, OC, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer69 YVMESMTYLUrinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer 70FLFPAFLTA AML, Gallbladder Cancer, Bile Duct Cancer 71 SLFPYVVLIMelanoma 72 SLDGNPLAVEsophageal Cancer, Uterine Cancer, Gallbladder Cancer, Bile Duct Cancer73 YIDPYKLLPL CLL, NHL, AML, Melanoma 74 SLTSFLISLUrinary bladder cancer 75 ALASAPTSVBRCA, Esophageal Cancer, Uterine Cancer, Gallbladder Cancer,Bile Duct Cancer 76 ILFDEVLTFACLL, NHL, AML, BRCA, Uterine Cancer, Gallbladder Cancer,Bile Duct Cancer 77 SLRAFLMPI AML 78 VLYGDVEELCLL, Urinary bladder cancer, Gallbladder Cancer, Bile Duct Cancer 79GLHQDFPSVVL NHL, BRCA, Uterine Cancer 81 VLAENPDIFAVSCLC, MCC, Melanoma, Urinary bladder cancer 82 VLDINDNPPV Melanoma 83QLLQYVYNL SCLC, NHL, AML, Uterine Cancer 85 QLIEKITQVSCLC, NHL, Melanoma, Urinary bladder cancer, GallbladderCancer, Bile Duct Cancer 86 SLQERQVFL NHL, Urinary bladder cancer 87ALPEPSPAA RCC, NHL, Melanoma, Gallbladder Cancer, Bile Duct Cancer 88LMAPAPSTV Brain Cancer, Esophageal Cancer, Urinary bladder cancer 89VLDEGLTSV SCLC, RCC, CLL, NHL, BRCA, Melanoma, Urinary bladder cancer,Uterine Cancer 90 TLNDGVVVQVRCC, CLL, NHL, Esophageal Cancer, Urinary bladder cancer, Uterine Cancer91 MLFENMGAYTV SCLC, CLL, NHL, Uterine Cancer cancer, NSCLC = non-smallcell lung cancer, SCLC = small cell lung cancer, RCC = kidney cancer,CRC =  colon or rectum cancer, GC = stomach cancer, HCC = liver cancer,PC = pancreatic cancer, PrC =  prostate cancer, leukemia, BRCA = breastcancer, OC = ovarian cancer, NHL = non-Hodgkin lymphoma, AML = acutemyelogenous leukemia, CLL = chronic lymphatic leukemia

TABLE 4B Peptides according to the present invention andtheir specific uses in other proliferativediseases, especially in other cancerous diseases.The table shows for selected peptides on whichadditional tumor types they were found and eitherover-presented on more than 5% of the measuredtumor samples, or presented on more than 5% ofthe measured tumor samples with a ratio ofgeometric means tumor vs normal tissues beinglarger than 3. Over-presentation is defined ashigher presentation on the tumor sample ascompared to the normal sample with highestpresentation. Normal tissues against whichover-presentation was tested were: adipose tissue,adrenal gland, artery, bone marrow, brain,central nerve, colon, esophagus, eye, gallbladder,heart, kidney, liver, lung, lymph node, whiteblood cells, pancreas, parathyroid gland,peripheral nerve, peritoneum, pituitary, pleura,rectum, salivary gland, skeletal muscle, skin,small intestine, spleen, stomach, thymus, thyroidgland, trachea, ureter, urinary bladder, vein. SEQ ID No SequenceAdditional Entities  2 PVCPPGGIQEV Esophageal Cancer  6 ALYDAELSQMEsophageal Cancer  8 AQLNIGNVLPV Urinary bladder cancer  9 STASAITPSVEsophageal Cancer 16 ALKDSVQRA HCC 43 AVLGGKLYV RCC, GC, HCC 45GLIEWLENTV HCC 47 QLLEGELETL OC 50 ALSNVVHKV NSCLC 51 FLIPSIIFA OC 56FLQLETEQV OC 57 AILGFALSEA HCC 60 SLGNFKDDLL NSCLC 61 FVAGYIAGV HCC 65ALWGFFPVLL HCC 66 TLLSEIAEL HCC 73 YIDPYKLLPLBRCA, Urinary bladder cancer 75 ALASAPTSV HCC 88 LMAPAPSTV Melanoma 89VLDEGLTSV GC NSCLC = non-small lung cancer, HCC = liver cancer, BRCA = breast cancer, RCC = renal cell carcinoma, GC = gastric cancer, OC= ovarian cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 10, 17, 37, 42, 48, 57, 61, 67, 69, 70, 72, 75, 76,78, and 87 for the—in one preferred embodiment combined—treatment ofgallbladder cancer and/or bile duct cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 2, 20, 50, 60, 61, 63, and 64 for the—in one preferredembodiment combined—treatment of NSCLC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 2, 17, 45, 59, 61, 63, 67, 81, 83, 85, 89, and 91 forthe—in one preferred embodiment combined—treatment of SCLC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 2, 3, 4, 6, 7, 8, 9, 16, 19, 23, 27, 32, 39, 41, 42,46, 48, 49, 50, 52, 53, 54, 58, 60, 61, 62, 72, 75, 88, and 90 forthe—in one preferred embodiment combined—treatment of esophageal cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 3, 4, 7, 8, 15, 16, 19, 20, 21, 24, 27, 31, 32, 39,45, 46, 47, 48, 50, 51, 53, 56, 58, 60, 64, 69, 73, 74, 78, 81, 85, 86,88, 89, and 90 for the—in one preferred embodiment combined—treatment ofurinary bladder cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 4, 16, 17, 39, 48, 50, 56, 57, 58, 62, 73, 75, 76, 79,and 89, for the—in one preferred embodiment combined—treatment of BRCA.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 8, 9, 11, 16, 17, 32, 43, 48, 56, 57, 65, 66, 67, 71,73, 81, 82, 85, 87, 88, and 89 for the—in one preferred embodimentcombined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 16, 17, 24, 29, 43, 57, 66, 70, 73, 76, 77, and 83 forthe—in one preferred embodiment combined—treatment of AML.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 16, 17, 19, 31, 43, 46, 47, 48, 49, 51, 56, 57, 58,66, 67, 72, 75, 76, 79, 83, 89, 90, and 91 for the—in one preferredembodiment combined—treatment of uterine cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 18, 58, 61, and 88 for the—in one preferred embodimentcombined—treatment of brain cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 32, 43, 48, 59, 60, 61, 63, 66, 73, 76, 79, 83, 85,86, 87, 89, 90, and 91 for the—in one preferred embodimentcombined—treatment of NHL.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 43, 61, 65, 66, 73, 76, 78, 89, 90, and 91 for the—inone preferred embodiment combined—treatment of CLL.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 45, 47, 51, 56, 66, and 67 for the—in one preferredembodiment combined—treatment of OC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 43, 50, 89, and 61 for the—in one preferred embodimentcombined—treatment of GC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 16, 43, 45, 57, 61, 65, 66, and 75 for the—in onepreferred embodiment combined—treatment of HCC.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 43, 61, 62, 87, 89, and 90 for the—in one preferredembodiment combined—treatment of RCC.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofhead and neck squamous cell carcinoma, acute myelogenous leukemia,breast cancer, bile duct cancer, brain cancer, chronic lymphocyticleukemia, colorectal carcinoma, esophageal cancer, gallbladder cancer,gastric cancer, hepatocellular cancer, melanoma, non-Hodgkin lymphoma,non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostatecancer, renal cell cancer, small cell lung cancer, urinary bladdercancer and uterine cancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) Class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 91.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (li), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 91, preferably containing SEQ IDNo. 1 to SEQ ID No. 31, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are head and neck squamous cellcarcinoma, acute myelogenous leukemia, breast cancer, bile duct cancer,brain cancer, chronic lymphocytic leukemia, colorectal carcinoma,esophageal cancer, gallbladder cancer, gastric cancer, hepatocellularcancer, melanoma, non-Hodgkin lymphoma, non-small cell lung cancer,ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer,small cell lung cancer, urinary bladder cancer, uterine cancer, andpreferably head and neck squamous cell carcinoma cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably head and necksquamous cell carcinoma. The marker can be over-presentation of thepeptide(s) themselves, or over-expression of the corresponding gene(s).The markers may also be used to predict the probability of success of atreatment, preferably an immunotherapy, and most preferred animmunotherapy targeting the same target that is identified by thebiomarker. For example, an antibody or soluble TCR can be used to stainsections of the tumor to detect the presence of a peptide of interest incomplex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of the inventive noveltargets in the context of cancer treatment.

ALOX12B is involved in terminal skin differentiation and epidermalbarrier function (Furstenberger et al., 2007; Epp et al., 2007). ALOX12Bis an immunosuppressive factor amplified in cancer (Rooney et al.,2015). Cisplatin induces and ATM phosphorylates (p)-DeltaNp63alpha.Subsequently, it up-regulates miR-185-5p which down-regulates let7-5presulting in the modulation of ALOX12B expression in squamous cellcarcinoma (Ratovitski, 2013). ALOX12B is associated with breast cancerand lung cancer risk (Lee et al., 2009; Shen et al., 2009).

ANKRD17 mRNA levels are widely down-regulated in colorectal carcinomasand make this protein a potential marker for multi-target assay panelsfor colorectal cancer detection (Ioana et al., 2010). Through thephosphorylation of ANKRD17 by cyclin E/CdK2 the protein is involved incell cycle regulation. Over-expression promotes S phase entry, whereasdepletion of expression inhibits DNA replication, blocks cell cycleprogression and up-regulates the expression of the tumor suppressors p53and p21 (Deng et al., 2009).

ATP5G1 is enriched in the oxidative phosphorylation pathway in gastriccancer (Song et al., 2016). ATP5G1 expression is reduced in head andneck squamous cell carcinoma (Koc et al., 2015). Knock-out of augmenterof liver regeneration (ALR) results in accelerated development ofsteatohepatitis and hepatocellular carcinoma in mice. Further, ATP5G1expression is reduced in ALR knock-down mice (Gandhi et al., 2015).

ATP5G2 is more methylated in high-grade bladder cancer tumors comparedto low-intermediate-grade tumors (Kitchen et al., 2016). RIZ1 is a tumorsuppressor whose depletion results in altered ATP5G2 expression (Xie etal., 2016b). ATP5G2 is highly expressed upon estrogen and progesteronetreatment in endometrial Ishikawa cancer cell line (Tamm-Rosenstein etal., 2013). ATP5G2 promoter is methylated in primary renal cellcarcinoma (Morris et al., 2011).

ATP5G3 is enriched in the oxidative phosphorylation pathway in gastriccancer (Song et al., 2016). ATP5G3 is up-regulated upon PPARalphaactivation which is negatively correlated with tumor progression andwhich suppresses cell migration (Huang and Chang, 2016). ATP5G3 may be aradiation susceptibility gene (Tsuji et al., 2005).

BTBD11 encodes BTB domain containing 11 and is located on chromosome12q23.3 (RefSeq, 2002). BTBD11 is differentially expressed in papillarythyroid cancer (Qu et al., 2016). BTBD11 is a TGF-beta target gene(Sawada et al., 2016). BTBD11 is mutated in gastric cancer (Leiserson etal., 2015; Leiserson et al., 2016).

Prognostic association of CD276 differs between smoking and non-smokingpatients with lung adenocarcinoma. High CD276 expression is linked tosmoking (Inamura et al., 2017). CD276 is hyper-methylated in prostatecancer (Wang et al., 2016b). CD276 is regulated by miR-124 which isdown-regulated in osteosarcoma. TGF-beta1 up-regulates miR-155 throughSMAD 3 and 4 signaling resulting in miR-143 attenuation by CEBPBinhibition which causes CD276 accumulation. CD276 is regulated bymiR-187 which is down-regulated in colorectal cancer (Wang et al.,2016e; Trojandt et al., 2016; Zhou et al., 2016; Wang et al., 2016a).CD276 mediates abnormal lipid metabolism in lung cancer by affectingSREBP-1/FASN signaling. Soluble CD276 mediates pancreatic cancerinvasion and metastasis through TLR4/NF-kappaB signaling (Xie et al.,2016a; Luo et al., 2016). CD276 is an immune checkpoint which may be apromising target in cancer therapy. It may be targeted during tumorgrowth to suppress anti-tumor immunity providing immune evasion for theemerging tumor (Leung and Suh, 2014; Swatler and Kozlowska, 2016;Janakiram et al., 2016). CD276 is expressed by most high-riskneuroblastomas, is over-expressed in tumor vasculature and plays animportant role in tumor survival and invasiveness (Bottino et al.,2014). Knock-down of CD276 increases chemo sensitivity and decreasesmetastatic potential. Knock-down of CD276 results in increased apoptoticmarker expression and STAT3 phosphorylation. Astragaloside IV treatmentreduces cell growth and increases chemo sensitivity to cisplatin byinhibiting CD276 in non-small cell lung cancer cells (Nygren et al.,2011; He et al., 2016). CD276 is over-expressed in esophageal cancer,breast cancer, gallbladder cancer, prostate cancer, and ovarian cancer(Barach et al., 2011; Janakiram et al., 2012; Fauci et al., 2012; Chenet al., 2016; Liu et al., 2016). CD276 over-expression is correlatedwith poor survival, prognosis, and tumor grade. CD276 promotes cancerinvasion and progression. However, CD276 may also have anti-tumoreffects. High CD276 expression is an indicator of lymph node metastasisand advanced TNM stage in non-small cell lung cancer (Yi and Chen, 2009;Loos et al., 2010; Nygren et al., 2011; Fauci et al., 2012; Wang et al.,2014a; Ye et al., 2016; Benzon et al., 2016; Wu et al., 2016). CD276down-regulates natural killer cells cytotoxicity supporting cancerimmune evasion (Bottino et al., 2014).

CDH23 encodes cadherin related 23 which is a member of the cadherinsuperfamily, whose genes encode calcium dependent cell-cell adhesionglycoproteins. The encoded protein is thought to be involved instereocilia organization and hair bundle formation. The gene is locatedin a region containing the human deafness loci DFNB12 and USH1D. Ushersyndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 arecaused by allelic mutations of this cadherin-like gene. Up-regulation ofthis gene may also be associated with breast cancer (RefSeq, 2002).TMPRSS3 is a poor prognostic factor in breast cancer and can interactwith CDH23 (Rui et al., 2015). CDH23 is up-regulated upon leptintreatment in ERalpha expressing breast cancer cells (Binai et al.,2013). CDH23 is up-regulated in breast cancer and may be involved inearly stage metastasis (Apostolopoulou and Ligon, 2012). Loss of CDH23can be observed in pancreatic cancer cell lines (Suzuki et al., 2008).

CDH3 is involved in oncogenic signaling and activates integrins,receptor tyrosine kinases, small molecule GTPases, EMT transcriptionfactors, and other cadherin family members. CDH3 signaling inducesinvasion and metastasis (Albergaria et al., 2011; Paredes et al., 2012;Bryan, 2015; Vieira and Paredes, 2015). Oncogenic activation of CDH3 isinvolved in gastric carcinogenesis (Resende et al., 2011). CDH3over-expression promotes breast cancer, bladder cancer, ovarian cancer,prostate cancer, endometrial cancer, skin cancer, gastric cancer,pancreas cancer, and colon cancer (Albergaria et al., 2011; Paredes etal., 2007; Bryan and Tselepis, 2010; Reyes et al., 2013; Vieira andParedes, 2015). CDH3 is a basal epithelial marker expressed inbasal-like breast cancer. BRCA1 carcinomas are characterized by theexpression of basal markers like CDH3 and show a high-grade, highlyproliferating, ER-negative, and HER3-negative phenotype (Honrado et al.,2006; Palacios et al., 2008; Rastelli et al., 2010; Dewar et al., 2011).CDH3 is a tumor suppressor in melanoma and oral squamous cell carcinoma(Haass et al., 2005; Vieira and Paredes, 2015). CDH3 may be used as EMTmarker. There is a shift from E-cadherin to N-cadherin and CDH3expression during tumor formation and progression (Piura et al., 2005;Bonitsis et al., 2006; Bryan and Tselepis, 2010; Ribeiro and Paredes,2014). Competitive interaction between CDH3 and beta-catenin causesimpaired intercellular interactions and metastases in gastric cancer(Moskvina and Mal'kov, 2010). CDH3 may be an early marker of cancerformation in colon cancer (Alrawi et al., 2006). Dys-regulation of CDH3is a marker for poor prognosis and increased malignancy (Knudsen andWheelock, 2005).

Over-expression of CLCA2 down-regulates beta-catenin andbeta-catenin-activated genes (Ramena et al., 2016). CLCA2 stronglyinteracts with EVA1 which is also inducible by p53 and p63, frequentlydown-regulated in breast cancer causing EMT, and important forepithelial differentiation. Both proteins interact with E-cadherin(Ramena et al., 2016). There is an AML1-CLCA2 and a RUNX1-CLCA2 genefusion product in adult acute myeloid leukemia (Giguere and Hebert,2010; Jiang et al., 2013). CLCA2 is inducible by p73, p53, and p63 uponDNA damage and acts as an inhibitor of proliferation (Walia et al.,2009; Sasaki et al., 2012; Yu et al., 2013; Ramena et al., 2016). CLCA2expression is elevated in circulating tumor cells from patients withlung adenocarcinoma and increased detection is associated with shortenedpatient survival (Hayes et al., 2006; Man et al., 2014). CLCA2 is higherexpressed in squamous cell carcinoma of the lung compared toadenocarcinoma and is associated with histological tumor grade. CLCA2expression may be used to detect non-small cell lung carcinoma and smallcell lung carcinoma (Hayes et al., 2006; Shinmura et al., 2014).Knock-down of CLCA2 causes epithelial-to-mesenchymal transition, cancercell migration, and invasion. Under normal condition, CLCA2 is thoughtto suppress migration and invasion through inhibition of the FAKsignaling pathway. CLCA2 mediates lung metastasis in association withbeta(4) integrin (Abdel-Ghany et al., 2001; Walia et al., 2012; Sasakiet al., 2012; Ramena et al., 2016). CLCA2 is down-regulated in breastcancer because of promotor hyper-methylation and is down-regulated incolorectal cancer. CLCA2 is differentially expressed in bladdercarcinoma and during the metastatic transformation of melanoma. Thereare copy number losses for CLCA2 in mantle cell lymphoma (Gruber andPauli, 1999; Bustin et al., 2001; Li et al., 2004; Balakrishnan et al.,2006; Riker et al., 2008; Walia et al., 2012; Matin et al., 2014; Ramenaet al., 2016).

DSG1 is over-expressed in keratocystic odontogenic tumors and shows highrates of expression in oral intra-epithelial neoplasms (Aizawa et al.,2014; Heikinheimo et al., 2015). DSG1 expression is lost in acantholyticsquamous cell carcinoma and down-regulated in chondrosarcoma, oralsquamous cell carcinoma, and lung cancer (Xin et al., 2014; Saaber etal., 2015; Galoian et al., 2015; Jurcic et al., 2015). DSG1 expressionis regulated by GRHL1 and GRHL1-negative mice treated with a standardchemical skin carcinogenesis protocol develop fewer papilloma but moresquamous cell carcinomas (Mlacki et al., 2014). DSG1, which is adownstream target of Rhoda and GEF Bcr, is a keratinocytedifferentiation marker (Dubash et al., 2013). KLK5 cleaves DSG1 whichmay be associated with metastases formation in oral squamous cellcarcinoma. Reduced levels of DSG1 might be involved in pancreatic cancerinvasion (Ramani et al., 2008; Jiang et al., 2011). Negative DSG1staining is associated with improved anal cancer-specific survival andpositive staining is associated with large tumor size and lymph nodemetastases. Loss of DSG1 is linked to bad prognosis in head and necksquamous cell carcinoma (Wong et al., 2008; Myklebust et al., 2012).Autoantibodies against DSG1 can be detected in paraneoplastic pemphigus(Seishima et al., 2004).

DSG3 expression in esophageal squamous cell carcinoma was shown to behighly correlated with histological grade and to have an impact onsurvival in esophageal squamous cell carcinoma, with negative DSG3expression indicating worse survival. Thus, DSG3 may be involved in theprogression of esophageal squamous cell carcinoma and may serve as aprognostic marker (Fang et al., 2014). In primary lung tumors, higherexpression of DSG3 and DSG2 was shown to be correlated to the diagnosisof squamous cell lung carcinoma, while a lower expression of DSG3 wasshown to be significantly linked to higher tumor grade. Thus, DSG3 mayserve as a potential diagnostic marker for squamous cell lung carcinomaand a potential differentiation marker for lung cancer (Saaber et al.,2015). DSG3 was described as a negative prognostic biomarker in resectedpancreatic ductal adenocarcinoma as high DSG3 expression was shown to beassociated with poor overall survival and poor tumor-specific survival.Thus, DSG3 and its downstream signaling pathways may be possibletherapeutic targets in DSG3-expressing pancreatic ductal adenocarcinoma(Ormanns et al., 2015).

Reduced expression of DSP is correlated with the tumor progression ofseveral cancers including breast, lung and cervical cancer(Schmitt-Graeff et al., 2007; Davies et al., 1999; Yang et al., 2012b).Expression of DSP significantly suppresses cell proliferation,anchorage-independent growth, migration and invasion in lung cancercells through inhibiting the Wnt/beta-catenin signaling pathway (Yang etal., 2012b).

DST may be related to breast cancer metastasis (Sun et al., 2006).Autoantibodies against DST can be found in lymphocytic leukemia andfollicular lymphomas (Aisa et al., 2005; Taintor et al., 2007). DST isup-regulated in 5-8F cells (high tumorigenic and metastatic ability) incomparison to 6-10B cells (tumorigenic, but lacking metastatic ability)in nasopharyngeal carcinoma (Fang et al., 2005). DST is highly expressedin head and neck squamous cell carcinoma (Lin et al., 2004). There areautoantibodies against DST in paraneoplastic pemphigus which isassociated with neoplasms (Yong and Tey, 2013; Wang et al., 2005; Preiszand Karpati, 2007; Zhu and Zhang, 2007). DST expression in prostatecancer is strongly inverse correlated with progression (Vanaja et al.,2003). Anti-DST autoantibodies are a promising marker for the diagnosisof melanoma (Shimbo et al., 2010). DST can be found in the urine ofcachectic cancer patients (Skipworth et al., 2010). DST isdifferentially expressed in adenocarcinomas and squamous cell carcinomasof the lung (McDoniels-Silvers et al., 2002). DST is distinctlyup-regulated with the onset of invasive cell growth (Herold-Mende etal., 2001).

EMC7 encodes ER membrane protein complex subunit 7 and is located onchromosome 15q14 (RefSeq, 2002). EMC7 may be a novel druggable targetand diagnostic biomarker in cancer (Delgado et al., 2014). EMC7 isdown-regulated in a pingyangmycin-resistant tongue squamous cellcarcinoma cell line (Zheng et al., 2010).

ESRP2 encodes an epithelial cell-type-specific splicing regulator(RefSeq, 2002). ESRP2 inhibits cancer cell motility in different cancertypes including lung and breast cancer cells. ESRP2 is down-regulated byTGF-beta in invasive fronts, leading to an increased expression ofepithelial-mesenchymal transition-associated transcription factors(Gemmill et al., 2011; Horiguchi et al., 2012; Ishii et al., 2014).

A mutation in the PH-domain binding motif of F2RL1 is sufficient todecrease breast tumor growth (Bar-Shavit et al., 2016). F2RL1 isover-expressed in gastric cancer and is inversely correlated withoverall survival of patients (Sedda et al., 2014). Tryptase is amediator of angiogenesis released by mast cells which activates F2RL1resulting in cancer cell proliferation, invasion, and metastasis (Marechet al., 2014; Ammendola et al., 2014). F2RL1 is affected bydifferentially expressed and mutated genes in cancer (D'Asti et al.,2014). F2RL1 is involved in cancer progression, invasion, and metastasis(Wojtukiewicz et al., 2015; Canto et al., 2012; Lima and Monteiro, 2013;Gieseler et al., 2013). F2RL1 is expressed in adenocarcinomas,melanomas, osteosarcomas, glioblastomas, meningiomas, leukemias, andsquamous cell carcinomas (Elste and Petersen, 2010). F2RL1 regulates theexpression of ALK5 which is a TGF-beta type I receptor. F2RL1 activatesMAP kinases (Oikonomopoulou et al., 2010; Witte et al., 2016).Up-regulation of tissue factor and integrins mediate F2RL1 signalingpromoting metastasis (Kasthuri et al., 2009; Ruf et al., 2011; Kocaturkand Versteeg, 2012; Ruf, 2012; Kocaturk and Versteeg, 2013). Trypsin andPAR2 form an autocrine loop promoting proliferation, invasion, andmetastasis. Trypsin stimulation may result in MAPK-ERK pathwayactivation through MMP- and PAR2-dependent activation of epidermalgrowth factor receptor (Soreide et al., 2006).

FAM160A1 encodes family with sequence similarity 160 member A1 and islocated on chromosome 4q31.3 (RefSeq, 2002). FAM160A1 expression isaltered upon DY131 binding to estrogen related receptor beta in prostatecancer (Lu et al., 2015b). There is a NFKB-FAM160A1 gene fusion productin prostate cancer (Teles, I et al., 2015). FAM160A1 is up-regulated inovarian cancer compared to benign tumors (Li et al., 2012a). A deletionin FAM160A1 can be found in familial and early-onset breast cancer(Krepischi et al., 2012). FAM160A1 is down-regulated in colorectalcancer (Li et al., 2012b).

FANCE is associated with esophageal squamous cell carcinoma risk (Li etal., 2013). Rare down-regulation of FANCE can be observed in head andneck squamous cell carcinoma (Wreesmann et al., 2007). Chkl mediates thephosphorylation of FANCE upon DNA crosslinking (Wang et al., 2007).Familial colorectal cancer shows a heterozygous genotype for FANCE.Fanconi anemia DNA damage repair may be linked to inheritedpre-disposition to colorectal cancer. An indel mutation might beinvolved in inherited esophageal squamous cell carcinoma. A missensevariant in FANCE was found in one family with breast cancer (Akbari etal., 2011; Seal et al., 2003; Esteban-Jurado et al., 2016). FANCE isinvolved in the regulation of cisplatin sensitivity (Taniguchi et al.,2003).

FAT1 was described as being significantly mutated in squamous-cellcancer of the head and neck, frequently mutated in cervicaladenocarcinoma, bladder cancer, early T-cell precursor acutelymphoblastic leukemia, fludarabine refractoriness chronic lymphocyticleukemia, glioblastoma and colorectal cancer and mutated in esophagealsquamous cell carcinoma (Gao et al., 2014; Neumann et al., 2013; Morriset al., 2013; Messina et al., 2014; Mountzios et al., 2014; Cazier etal., 2014; Chung et al., 2015). FAT1 was described as being repressed inoral cancer and preferentially down-regulated in invasive breast cancer(Katoh, 2012). FAT1 was described as being up-regulated in leukemiawhich is associated with a poor prognosis in preB-acute lymphoblasticleukemia (Katoh, 2012).

FAT1 was shown to be up-regulated in pancreatic adenocarcinoma andhepatocellular carcinoma (Valletta et al., 2014; Wojtalewicz et al.,2014). FAT1 was described to suppress tumor growth via activation ofHippo signaling and to promote tumor migration via induction of actinpolymerization (Katoh, 2012). FAT1 was shown to be a candidate cancerdriver gene in cutaneous squamous cell carcinoma (Pickering et al.,2014). FAT1 was described as a tumor suppressor which is associated withWnt signaling and tumorigenesis (Morris et al., 2013).

FAT3 was shown to be down-regulated in Taxol resistant ovarian carcinomacell lines upon silencing of androgen receptor, resulting in increasedsensitization to Taxol in these cell lines. Thus, FAT3 may be acandidate gene associated with Taxol resistance (Sun et al., 2015b).FAT3 was shown to be mutated in esophageal squamous cell carcinoma,resulting in dysregulation of the Hippo signaling pathway (Gao et al.,2014). FAT3 was shown to be mutated recurrently in early T-cellprecursor acute lymphoblastic leukemia (Neumann et al., 2013). FAT3 wasdescribed as a gene with signatures specific for meningothelialmeningiomas, therefore being associated with tumorigenesis in thissubtype of benign meningiomas (Fevre-Montange et al., 2009). FAT3 wasdescribed as a tumor suppressor which is repressed upon lung cancerdevelopment from dysplastic cells (Rohrbeck and Borlak, 2009).

FHAD1 is down-regulated upon NFE2 knock-down which is involved inoxidative stress response (Williams et al., 2016). CpG methylation ofFHAD1 may be used as biomarker in metastatic-lethal prostate cancer(Zhao et al., 2017). FHAD1 is down-regulated in esophageal squamous cellcarcinoma and may contribute to chemoresistance against cisplatin(Tsutsui et al., 2015). FHAD1 may be a tumor suppressor gene in breastcancer (Iorns et al., 2012).

G3BP1 encodes G3BP stress granule assembly factor 1 which is one of theDNA-unwinding enzymes which prefers partially unwound 3′-tailedsubstrates and can also unwind partial RNA/DNA and RNA/RNA duplexes inan ATP-dependent fashion (RefSeq, 2002). G3BP1 may be used as biomarkerfor drug response in HER2+ breast cancer (Chien et al., 2016).miR-193a-3p, which inhibits the progression and metastasis of lungcancer in vitro and in vivo, down-regulates G3BP1 (Deng et al., 2015).G3BP1 is a direct target of resveratrol. Depletion of G3BP1 reduces theresveratrol-induced p53 expression and apoptosis. G3BP1 is a negativeregulator of p53 by interacting with USP10, a p53-specificdeubiquitinase (Oi et al., 2015). G3BP1 is recruited by MYCNOS to thepromotor region of MYCN to regulate its expression. G3BP1 negativelyregulates PMP22 to increase proliferation in breast cancer (Winslow etal., 2013; Vadie et al., 2015). G3BP1 may be the target of abi-functional peptide consisting of a cancer recognition peptide and apro-apoptotic peptide (Meschenmoser et al., 2013). Late-stage oralsquamous cell carcinomas are sensitive to G3BP1 knock-down causingincreased apoptosis (Xu et al., 2013). G3BP1 is up-regulated in breastcancer, oral squamous cell carcinoma, colon cancer, pancreas cancer,hepatocellular carcinoma and gastric cancer and is correlated with poorpatient prognosis, tumor size, vascular invasion, T classification,lymph node metastasis, TNM stage, and reduced overall survival (Lo etal., 2012; Winslow et al., 2013; Min et al., 2015; Dou et al., 2016).Y-box binding protein 1 binds to the 5′UTR of G3BP1 mRNA to regulate theavailability of the G3BP1 stress granule nucleator for stress granuleassembly. Down-regulation of either YB-1 or G3BP1 results in reducedstress granule formation and tumor invasion (Ward et al., 2011;Annibaldi et al., 2011; Somasekharan et al., 2015). G3BP1 controls theactivity of the H+-ATPase and the translation of beta-F1-ATPase mRNA(Willers and Cuezva, 2011). G3BP1 co-localizes with promyelocyticleukemia nuclear bodies before and after ionizing radiation (Liu et al.,2010). Epigallocatechin gallate, the major compound of green tea,suppresses lung tumorigenesis by binding to G3BP1. G3BP1 expression isaffected by lovostatin (Klawitter et al., 2010; Shim et al., 2010).G3BP1 is involved in cancer cell growth, apoptosis, motility, migration,invasion, and metastasis by up-regulating Slug. Knock-down of G3BP1decreases Slug expression and increases the epithelial markerE-cadherin. Up-regulation of G3BP1 in breast cancer activatesepithelial-to-mesenchymal transition via the Smad signaling pathway.G3BP1 is involved in Ras and NF-kappaB signaling, ubiquitin proteasomepathway, and RNA processing (French et al., 2002; Zhang et al., 2015;Dou et al., 2016). Forced G3BP1 expression promotes cell migration inhepatocellular carcinoma (Dou et al., 2016).

GAR1 is able to activate p53 (Zhang et al., 2012). GAR1 is involved inthe telomerase complex (Zhu et al., 2004; Rashid et al., 2006; Tomlinsonet al., 2008; Pigullo et al., 2009; Low and Tergaonkar, 2013;Heidenreich et al., 2014). GAR1 is important for cell viability (Lubbenet al., 1995).

ITGB4 is associated with prostate cancer, gastric cancer, breast cancer,oral squamous cell carcinoma and ovarian cancer and was shown to beup-regulated in pancreatic ductal adenocarcinoma (Chen et al., 2014; Xinet al., 2014; Zubor et al., 2015; Masugi et al., 2015; Gao et al., 2015;Kawakami et al., 2015). ITGB4 (also called CD104) tends to associatewith the alpha 6 subunit and is likely to play a pivotal role in thebiology of several invasive carcinoma such as esophageal squamous cellcarcinoma, bladder and ovarian carcinoma (Kwon et al., 2013; Pereira etal., 2014; Chen et al., 2014). A single nucleotide polymorphism in ITGB4seems to influence tumor aggressiveness and survival and may haveprognostic value for breast cancer patients (Brendle et al., 2008).

KDM5B encodes the protein JARID1B, a lysine-specific histone demethylasethat is capable of repressing certain tumor suppressor genes byde-methylating lysine 4 of histone H3 (RefSeq, 2002). As epigeneticfactor, KDM5B supports proliferation, migration and invasion of humanOSCC, head and neck squamous cell carcinoma (HNSCC), breast cancer andlung cancer by suppressing p53 expression (Shen et al., 2015; Tang etal., 2015a; Zhao and Liu, 2015; Lin et al., 2015). Also, known as JARID1B, KDM5B promotes metastasis an epithelial-mesenchymal transition invarious tumor types via PTEN/AKT signaling (Tang et al., 2015a).

KLHL21 is up-regulated in hepatocellular carcinoma and may be used asbioclinical marker (Shi et al., 2016). KLHL21 is a negative regulator ofIKKbeta. KLHL21 expression is down-regulated in macrophages uponpro-inflammatory stimuli. Over-expression of KLHL21 inhibits IKKbetaactivation and IkappaBalpha degradation (Mei et al., 2016). KLHL21 isover-expressed by the aberrant gene fusion transcription factorASPSCR1-TFE3 which is found in two distinct entities, alveolar soft partsarcoma and renal cell carcinoma (Kobos et al., 2013). KLHL21 may beinvolved in cancer (Martinez et al., 2010). KLHL21 is necessary forcytokinesis and regulates chromosomal passenger complex translocationfrom chromosomes to the spindle midzone in anaphase. It interacts withthe Cullin3-based E3 ubiquitin ligase and directly binds to Aurora Bcausing its ubiquitination (Maerki et al., 2009). KLHL21 negativelyregulates TNF alpha-activated NF-kappa B signaling via targeting IKKbeta (Mei et al., 2016).

KLK6 encodes kallikein related peptidase 6 which is a member of thekallikrein subfamily of the peptidase S1 family of serine proteases.Growing evidence suggests that many kallikreins are implicated incarcinogenesis and some have potential as novel cancer and other diseasebiomarkers. Expression of this protease is regulated by steroid hormonesand may be elevated in multiple human cancers and in serum frompsoriasis patients. The encoded protease may participate in the cleavageof amyloid precursor protein and alpha-synuclein, thus implicating thisprotease in Alzheimer's and Parkinson's disease, respectively. This geneis located in a gene cluster on chromosome 19 (RefSeq, 2002). KLK6 isinducible by p53 and its expression increased autophagy and drugresistance in gastric cancer (Kim et al., 2016). Down-regulation of KLK6is associated with increased GNA13 expression which is linked toinvasiveness of benign breast tumors (Teo et al., 2016). KLK6 is able toup- and down-regulate several miRNAs, which may affect cell cycle, MYC,MAPK, and other signaling pathways (Sidiropoulos et al., 2016). KLK6belongs to a set which is linked to panitumumab resistance in metastaticcolorectal cancer (Barry et al., 2016). KLK6 is associated with theregulation of axonal growth following spinal injury, tumor cellmetastasis, and alpha synuclein aggregate pathologies like Parkinson'sdisease (Xi et al., 2015). KLK6 is over-expressed in highly invasive PC3prostate cancer and ovarian cancer and dys-regulated in cervicalpre-cancer (Tamir et al., 2014; Hwang and Lindholm, 2015). KLK6 may beused as biomarker in a variety of entities including hepatocellularcarcinoma, breast cancer, colon cancer, gastrointestinal cancer, andastrocytoma (Vakrakou et al., 2014; Yu et al., 2015b; Grin et al., 2015;Schrader et al., 2015; Drucker et al., 2015; Mange et al., 2016). KLK6is associated with overall survival in advanced serous ovarian cancerand its expression might be linked to other clinical parameters (Kolinet al., 2014; Dorn et al., 2015; Yang et al., 2016a; Leung et al., 2016;Ahmed et al., 2016).

Knock-down of CD34, whose expression in head and neck squamous cellcarcinoma is associated with cell cycle progression, up-regulates KRT1expression (Ettl et al., 2016). KRT1 is down-regulated in HepG2 cellsupon platycodin D treatment (Lu et al., 2015a). Immunohistochemicalstaining of a clear cell focus from a Bowens disease superficialinvasive carcinoma is negative for KRT1 (Misago et al., 2016). miR-944induces KRT1 expression by up-regulation of p53 and impairing of ERKsignaling (Kim et al., 2015). KRT1 expression is up-regulated in earlyand late stage of squamous cell carcinoma (Tang et al., 2015b). Nucleardegradation down-regulates KRT1 expression (Naeem et al., 2015). KRT1expression may be a marker for differentiation status. In combinationwith NMP-52 and AFP expression, it may be used to detect hepatocellularcarcinoma (Attallah et al., 2015; Bruna et al., 2017). KRT1 isup-regulated upon docosahexaenoic acid treatment, which is known toreduce breast cancer invasion (Blanckaert et al., 2015). Proliferatingbasophilic cells from onychocytic carcinoma failed to express KRT1 (Wanget al., 2015a). Up-regulation of KRT1 is indirectly associated withNotch1 receptor stimulation (Vliet-Gregg et al., 2015). S100A7down-regulates KRT1 (Li et al., 2015). Expression of KRT1 is associatedwith p21 and hsp70 expression in oral squamous cell carcinoma. KRT1absence is correlated with the absence of Klf4, which is a transcriptionfactor that suppresses cell proliferation and promotes differentiation(Paparella et al., 2015; Frohwitter et al., 2016).

KRT13 encodes keratin 13 which is a member of the keratin gene family.Vitamin D alters KRT13 expression (Narayanan, 2006). Immunostaining forCK13 is positive in the epidermoid component of mucoepidermoid carcinomaand is negative in canalicular adenoma and oncolytic carcinoma arisingin submandibular gland (Muramatsu et al., 2003; Matsuzaka et al., 2004;do Prado et al., 2007). Aberrant expression of alpha 6 beta 4 integrinup-regulates KRT13, an early event in the development of squamous cancerof the skin (Tennenbaum et al., 1996). KRT13 may be used as biomarkerfor cervical intraepithelial neoplasia. Loss of KRT13 is a marker fortumor grade and stage in transitional urothelial cell carcinoma. KRT13expression is a marker for skin cancer progression (Slaga et al., 1995;Southgate et al., 1999; Duggan, 2002; Baak et al., 2006).

KRT13 expression is down-regulated in oral cancer stem cells and oralsquamous cell carcinoma (Morgan and Su, 1994; Sinha et al., 2013). KRT14was highly expressed in various squamous cell carcinomas such asesophageal, lung, larynx, uterine cervical as well as in adenomatoidodontogenic tumor. However, it was absent in small cell carcinoma of theurinary bladder and weak in lung adenocarcinoma, gastric adenocarcinoma,colorectal adenocarcinoma, hepatocellular carcinoma, pancreatic ductaladenocarcinoma, breast infiltrating dutal adenocarcinoma, thyroidpapillary carcinoma and uterine endometrioid adenocarcinoma (Xue et al.,2010; Terada, 2012; Vasca et al., 2014; Hammam et al., 2014; Shruthi etal., 2014). In bladder cancer, KRT14 expression was strongly associatedwith poor survival (Volkmer et al., 2012).

Long-time exposure of MCF-7 breast cancer cells to ethanol up-regulatesKRT15 which is a malignancy-related gene (Gelfand et al., 2017). Basalcell carcinomas with invasive growth show a negative expression of KRT15(Ziari et al., 2015). KRT15 may be used to discriminate spiradenomas andcylindromas (Sellheyer, 2015). KRT15 is sequentially up-regulated inbladder carcinogenesis (Chuang et al., 2014). SIRT2, which isdown-regulated in skin cancer, inhibits KRT15 expression, which is anepithelial stem cell marker (Wang et al., 2014b). KRT15 is a hairfollicle stem cell marker (Bongiovanni et al., 2014; Koba et al., 2015;Narisawa et al., 2015). KRT15 is an undifferentiated basal cell markermore strongly expressed in malignant compared to benign ocular surfacesquamous neoplasia (Nagata et al., 2014). Spheroid-selected epidermalsquamous cell carcinomas have an enriched KRT15 expression (Adhikary etal., 2013). KRT15 is up-regulated in urothelial carcinoma (Tai et al.,2013). KRT15 staining of actinic keratoses associated with head and necktumors is positive in 7% of the cases and in 36% in adenoid cysticcarcinoma. Staining is higher in desmoplastic trichoepithelioma comparedto morphea form basal cell carcinoma and microcystic adnexal carcinoma(Sabeti et al., 2013; Evangelista and North, 2015; North et al., 2015;Solus et al., 2016). KRT15 up-regulation affects overall survival innon-small cell lung cancer and may be used as marker for differentialdiagnosis of NSCLC (Gomez-Morales et al., 2013; Boyero et al., 2013).KRT15 is regulated by p53 and ER (Lion et al., 2013).

Over-expression of KRT16 was found in basal-like breast cancer celllines as well as in carcinoma in situ. Others did not find significantdifference in immunohistochemical expression of KRT16 betweennon-recurrent ameloblastomas and recurrent ameloblastomas (Joosse etal., 2012; Ida-Yonemochi et al., 2012; Safadi et al., 2016). Inaddition, in silico analyses showed correlation between KRT16 expressionand shorter relapse-free survival in metastatic breast cancer (Joosse etal., 2012).

KRT5 was shown to be up-regulated in breast cancers of young women(Johnson et al., 2015). KRT5 was shown to be associated with inferiordisease-free survival in breast cancer in young women and withunfavorable clinical outcome in premenopausal patients with hormonereceptor-positive breast cancer (Johnson et al., 2015; Sato et al.,2014). KRT5 was shown to be regulated by the tumor suppressor BRCA1 inthe breast cancer cell lines HCC1937 and T47D (Gorski et al., 2010).KRT5 was shown to be de-regulated in malignant pleural mesothelioma(Melaiu et al., 2015). KRT5 was described as a diagnostic mesothelialmarker for malignant mesothelioma (Arif and Husain, 2015). KRT5 wasshown to be correlated with the progression of endometrial cancer (Zhaoet al., 2013). KRT5 was shown to be mutated and down-regulated ininvasive tumor areas in a patient with verrucous carcinoma (Schumann etal., 2012). KRT5 was shown to be part of a four-protein panel which wasdifferentially expressed in colorectal cancer biopsies compared tonormal tissue samples (Yang et al., 2012a). KRT5 and three otherproteins of the four-protein panel were described as novel markers andpotential targets for treatment for colorectal cancer (Yang et al.,2012a). KRT5 was described as being associated with basal cell carcinoma(Depianto et al., 2010). KRT5 was described as a candidate to identifyurothelial carcinoma stem cells (Hatina and Schulz, 2012).

KRT6A was described as part of a seven-gene signature that could be usedas a prognostic model to predict distant recurrence-free survival inpatients with triple negative breast cancer who received adjuvantchemotherapy following surgery (Park et al., 2015b). KRT6A was shown tobe up-regulated in horn cancer in Bos indicus and in gastric cancer(El-Rifai et al., 2002; Koringa et al., 2013). KRT6A was shown to bedown-regulated in two cases of vulvar sarcomas with morphologic andimmunohistochemical characteristics of extraskeletal myxoidchondrosarcoma (Dotlic et al., 2014). KRT6A expression was shown to bealtered in oral squamous cell carcinoma (Chanthammachat et al., 2013).KRT6A was shown to be associated with negative regulation ofproto-oncogene Src kinase activity and the migratory potential of skinkeratinocytes during wound repair. This may be important in relatedcontexts such as cancer (Rotty and Coulombe, 2012). KRT6A was shown tomark mammary bi-potential progenitor cells that can give rise to aunique mammary tumor model resembling human normal-like breast cancer(Bu et al., 2011). KRT6A was described as an important part of a 25-genetranscriptional network signature which can be used to distinguishadenocarcinomas and squamous cell carcinomas of the lung (Chang et al.,2011).

KRT6B was shown to be a candidate for down-regulated genes in theesophageal cancer cell line KYSE170 (Kan et al., 2006). KRT6B was shownto be up-regulated in renal cell carcinoma, sporadic keratocysticodontogenic tumors and in horn cancer in Bos indicus (Koringa et al.,2013; Hu et al., 2015; Heikinheimo et al., 2007). KRT6B loss-of-functionwas shown to inhibit the expression of notch1 and induce renal cellcarcinoma cell death in vitro. Thus, KRT6B interaction with notch1 wasdescribed to contribute to progression of renal cell carcinoma (Hu etal., 2015). KRT6B was described as a basal-like breast cancer-associatedcytokeratin whose expression was diminished upon decreased expression ofbasal-like tumor-associated GABRP in the cell lines HCC1187 and HCC70(Sizemore et al., 2014). Selective inhibition of ERK1/2 was also shownto result in decreased expression of basal-like cytokeratins such asKRT6B and decreased migration in basal-like breast cancer (Sizemore etal., 2014). Thus, the GABRP-ERK1/2-cytokeratin axis is involved inmaintaining the migratory phenotype of basal-like breast cancer(Sizemore et al., 2014). KRT6B was shown to be associated with negativeregulation of proto-oncogene Src kinase activity and the migratorypotential of skin keratinocytes during wound repair. This may beimportant in related contexts such as cancer (Rotty and Coulombe, 2012).KRT6B was described as an important part of a 25-gene transcriptionalnetwork signature which can be used to distinguish adenocarcinomas andsquamous cell carcinomas of the lung (Chang et al., 2011). KRT6B wasshown to be differentially expressed during benzo (a) pyrene inducedtumorigenesis of human immortalized oral epithelial cells (Li et al.,2008).

KRT6C was described as an important part of a 25-gene transcriptionalnetwork signature which can be used to distinguish adenocarcinomas andsquamous cell carcinomas of the lung (Chang et al., 2011).

KRT75 encodes keratin 75 which is a member of the type II keratin familyclustered on the long arm of chromosome 12. The encoded protein plays anessential role in hair and nail formation. Variations in this gene havebeen associated with the hair disorders pseudofolliculitis barbae (PFB)and loose anagen hair syndrome (LAHS) (RefSeq, 2002). KRT75 isdown-regulated in an in vivo passaged and re-derived prostatic cell linecompared to the parental cell line (Sivanathan et al., 2014). KRT75 isexpressed in onychomatricoma which may suggest a differentiation towardthe nail bed and the nail isthmus (Perrin et al., 2011). KRT75 isdown-regulated in 21T breast cells (Xu et al., 2010). KRT75 expressioncan be changed by proteasome inhibitors and dexamethasone (Kinyamu etal., 2008).

Inhibition of LAP3 was shown to result in suppressed invasion in theovarian cancer cell line ES-2 through down-regulation of fascin andMMP-2/9. Thus, LAP3 may act as a potential anti-metastasis therapeutictarget (Wang et al., 2015b). High expression of LAP3 was shown to becorrelated with grade of malignancy and poor prognosis of gliomapatients (He et al., 2015). LAP3 was shown to promote glioma progressionby regulating cell growth, migration and invasion and thus might be anew prognostic factor (He et al., 2015). Frameshift mutations in genesinvolved in amino acid metabolism including LAP3 were detected inmicrosatellite instability-high gastric and colorectal cancer (Oh etal., 2014). LAP3 was shown to be up-regulated in hepatocellularcarcinoma, esophageal squamous cell carcinoma and prostate cancer (Zhanget al., 2014a; Tian et al., 2014; Lexander et al., 2005). LAP3 was shownto promote hepatocellular carcinoma cells proliferation by regulatingG1/S checkpoint in cell cycle and advanced cells migration (Tian et al.,2014). Expression of LAP3 was further shown to be correlated withprognosis and malignant development of hepatocellular carcinoma (Tian etal., 2014). Silencing of LAP3 in the esophageal squamous cell carcinomacell line ECA109 was shown to reduce cell proliferation and colonyformation while LAP3 knock-down resulted in cell cycle arrest (Zhang etal., 2014a). Over-expression of LAP3 in the esophageal squamous cellcarcinoma cell line TE1 was shown to favor cell proliferation andinvasiveness (Zhang et al., 2014a). Thus, LAP3 was shown to play a rolein the malignant development of esophageal squamous cell carcinoma(Zhang et al., 2014a).

High levels of LGALS7 are associated with an aggressive phenotype inbreast cancer with increased cancer invasiveness, growth, and metastasis(Grosset et al., 2016). LGALS7 is differentially expressed in the serumof colorectal cancer patients whereas immunohistochemical staining ofCRC tumors is negative for LGALS7 (Lim et al., 2016). LGALS7 isdown-regulated in prostate cancer, cervical cancer, and vulvar squamouscell carcinoma and is correlated with advanced clinical stage, poordifferentiation, and regional lymph node metastasis. Re-expression leadsto increased apoptosis. Increased promotor methylation is associatedwith advanced clinical stage, poor differentiation, and regional lymphnode metastasis in VSCC (Labrie et al., 2015; Jiang et al., 2015;Higareda-Almaraz et al., 2016). Cytoplasmic expressed LGALS7 inhibitsp53 and increases chemoresistance in breast cancer (Grosset et al.,2014). LGALS7 is not expressed in normal ovarian tissue and expressed inepithelial ovarian cancer. Expression is more frequent in high grade andmetastatic tumors and correlates with overall survival. LGALS7expression is induced by mutant p53 (Kim et al., 2013; Labrie et al.,2014). LGALS7 may be used as biomarker for metastatic cutaneousmelanoma. It is also correlated with clinical parameters in head andneck squamous and basal cell carcinoma (Timar et al., 2010; Cada et al.,2009). LGALS7 is related to breast cancer incidence (Tang et al., 2008).LGALS7 expression can be induced by p53 and is able to act pro-apoptotic(Ueda et al., 2004).

LGALS7B potentiates the HER2 positive breast cancer aggressiveness(Grosset et al., 2016). LGALS7B regulates molecules involved inapoptosis, tissue morphogenesis, metabolism, transport, chemokineactivity, and immune response (Higareda-Almaraz et al., 2016). LGALS7Bis down-regulated in cervical cancer. High LGALS7B expression inassociation with low Gal-1 expression is linked to better prognosis(Higareda-Almaraz et al., 2016). LGALS7B is hyper-methylated in vulvarsquamous cell carcinoma (Jiang et al., 2015). Re-expression of LGALS7Bin prostate cancer enhances chemosensitivity to etoposide and cisplatin(Labrie et al., 2015). LGALS7B is down-regulated in vulvar squamous cellcarcinoma, prostate cancer, and colorectal cancer. Down-regulation ofLGALS7B is associated with advanced clinical stage, poor tumordifferentiation, and regional lymph node metastasis (Labrie et al.,2015; Lim et al., 2016; Jiang et al., 2015). Cytosolic LGALS7B inhibitsdox-induced PARP-1 cleavage resulting in suppressed p53 activation anddecreased p21 and CDKN1A expression in breast cancer (Grosset et al.,2014). LGALS7B up-regulates apoptosis and inhibits IL-2 and IFN-gammaexpression (Yamaguchi et al., 2013). LGALS7B is over-expressed inovarian cancer and is associated with greater age, high mortality,increased tumor volume, and poor survival (Kim et al., 2013; Labrie etal., 2014). Immunohistochemical staining of LGALS7B may be used todistinguish salivary gland tumor types (Remmelink et al., 2011). LGALS7Bis down-regulated in head and neck basal cell carcinoma. In head andneck squamous cell carcinoma, LGALS7B shows different expressionpatterns and different expression levels are correlated withkeratinization and differentiation (Cada et al., 2009).

MALL may be used to categorize lung cancer subtypes (Watanabe et al.,2010). MALL may be a metastasis suppressor gene in prostate cancer (Yiet al., 2009). MALL mRNA and protein expression is reduced in coloncancer patients and is associated with vessin invasion, diseaserecurrence, and metastasis or death. Loss of MALL is associated withdecreased overall survival and disease-free survival. Over-expression ofMALL suppresses cell proliferation and inhibits migration in cell lines(Fan et al., 2011; Kim et al., 2008a; Wang et al., 2016c). MALL can befound on prostasomes secreted by a prostate cancer cell line where itinteracts with caveolin-1 (Llorente et al., 2004). MALL isdown-regulated in non-small cell lung cancer and cervical squamous cellcancer. It is differentially expressed in glioma cells (Ai et al., 2003;Hatta et al., 2004; Kettunen et al., 2004).

MCM4 expression is associated with up-regulated carbonic anhydrase IX, atransmembrane glycoprotein which is correlated with decreased survivaland cancer progression in several entities including esophageal cancer(Huber et al., 2015). Has-miR-615-3p may be involved in nasopharyngealcarcinoma by regulating MCM4 (Chen et al., 2015). MCM4 might play a rolein the development of bladder cancer (Zekri et al., 2015). Again-of-function mutant of p53 increases the expression of MCM4 inbreast cancer (Polotskaia et al., 2015). There is a mutation of MCM4 inhuman skin cancer which shows reduced DNA helicase activity (Ishimi andIrie, 2015). MCM4 over-expression alone is only weakly associated withshorter survival in breast cancer. Over-expression of all six parts ofthe MCM complex is strongly associated with shorter survival (Kwok etal., 2015). MCM4 is differentially expressed in lung adenocarcinoma andlaryngeal squamous cell carcinoma (Lian et al., 2013; Zhang et al.,2014b). MCM4 is significantly over-expressed in cervical cancer (Das etal., 2013; Das et al., 2015). MCM4 may be used as biomarker forcolorectal cancer (Fijneman et al., 2012).

Antizyme inhibitor inhibits ubiquitin-independent degradation of ODC1resulting in accelerated polyamine formation which triggers thedevelopment of gastric cancer, breast cancer, hepatocellular carcinoma,and esophageal squamous cell carcinoma (Qiu et al., 2016). Piroxicaminhibits ODC1-dependent polyamine production involved in non-melanomaskin carcinogenesis (Campione et al., 2015). ODC1 regulates putrescinewhich is important for cell division regulation, differentiation,maturation, and apoptosis (Ramani et al., 2014; Zdrojewicz andLachowski, 2014). ODC1 is a Myc and MYCN target gene and high ODC1expression is associated with reduced event-free survival inneuroblastoma (Funakoshi-Tago, 2012; Saletta et al., 2014). BlockingODC1 may be used for chemoprophylaxis in colorectal cancer (Zhou et al.,2012).

PARP9 (also known as ARTD9) encodes poly(ADP-ribose) polymerase familymember 9 and is located on chromosome 3q21.1 (RefSeq, 2002). DTX3L formscomplexes with ARTD8 and PARP9 promoting proliferation, chemoresistance,and survival of metastatic prostate cancer cells by inhibiting the tumorsuppressor IRF1 (Bachmann et al., 2014). PARP9 inhibitsIFN-gamma-STAT1-IRF1-p53 signaling in diffuse large B cell lymphoma andactivates the expression of the proto-oncogenes IRF2 and BCL-6. Thisresults in proliferation, survival, and chemoresistance in DLBCL(Camicia et al., 2013). PARP9 expression is IFN-gamma-inducible(Juszczynski et al., 2006). PARP9 may be a drug target in diffuse largeB cell lymphoma. PARP9 is an oncogenic survival factor in high-risk,chemoresistant DLBCL (Bachmann et al., 2014; Aguiar et al., 2005;Camicia et al., 2015). PKP1 was shown to be down-regulated in prostatecancer and esophageal adenocarcinoma (Kaz et al., 2012; Yang et al.,2015). Knock-down of PKP1 in the non-neoplastic, prostatic BPH-1 cellline led to reduced apoptosis and differential expression of genes suchas the prostate cancer-associated SPOCK1 gene (Yang et al., 2015).Collectively, altered expression of PKP1 and SPOCK1 appears to befrequent and critical event in prostate cancer and PKP1 is suggested tohave a tumor-suppressive function (Yang et al., 2015). Reducedexpression of PKP1 was shown to be associated with significantly shortertime to onset of distant metastasis in oral cavity squamous cellcarcinoma (Harris et al., 2015). PKP1 loss through promoter methylationwas described to be associated with the progression of Barrett'sesophagus to esophageal adenocarcinoma (Kaz et al., 2012). PKP1 wasshown to be up-regulated in non-small cell lung cancer and may be a goodmarker to distinguish squamous-cell carcinomas samples (Sanchez-Palenciaet al., 2011). PKP1 was shown to be up-regulated in thewell-differentiated liposarcoma cell line GOT3 (Persson et al., 2008).Decreased PKP1 expression was described to promote increased motility inhead and neck squamous cell carcinoma cells (Sobolik-Delmaire et al.,2007). PKP1 loss was shown to be associated with cervical carcinogenesis(Schmitt-Graeff et al., 2007). PKP1 was shown to be associated withlocal recurrences or metastases as well as poor survival in patientswith squamous cell carcinoma of the oropharynx (Papagerakis et al.,2003).

PLEC is over-expressed in colorectal adenocarcinoma, head and necksquamous cell carcinoma and pancreatic cancer (Lee et al., 2004; Katadaet al., 2012; Bausch et al., 2011).

Periplakin is decreased in T24CDDPR bladder cancer cells (Taoka et al.,2015). PPL staining is lower in bladder cancer compared to healthytissue. Loss of PPL is linked to pathological stage and survival(Matsumoto et al., 2014). PPL is correlated with epithelial-like tumorcells (Kohn et al., 2014). EVPL, periplakin, and involucrin-negativemice show a skin cancer-resistant phenotype (Cipolat et al., 2014;Natsuga et al., 2015; Natsuga et al., 2016). PPL is highly expressed intriple-negative breast cancer (Choi et al., 2013). Periplakin isdown-regulated in esophageal squamous cell carcinoma because ofhyper-methylation. PPL knock-down in ESCC is associated with reducedcellular movement and attachment (Otsubo et al., 2015; Tonoike et al.,2011). Paraneoplastic pemphigus shows autoantibodies against PPL (Yongand Tey, 2013; Li et al., 2009; Probst et al., 2009; Zimmermann et al.,2010).

PRKDC is a frequently mutated gene in endometriosis-associated ovariancancer and breast cancer (Er et al., 2016; Wheler et al., 2015). PRKDCis up-regulated in cancerous tissues compared with normal tissues incolorectal carcinoma. Patients with high PRKDC expression show pooreroverall survival (Sun et al., 2016).

PRNP encodes a membrane glycosylphosphatidylinositol-anchoredglycoprotein that tends to aggregate into rod-like structures andcontains a highly unstable region of five tandem octapeptide repeats.Mutations in the repeat region as well as elsewhere in this gene havebeen associated with various prion diseases. An overlapping open readingframe has been found for this gene that encodes a smaller, structurallyunrelated protein, AltPrp (RefSeq, 2002). Although its physiologicalrole is not completely defined, PRNP is involved in self-renewal,pluripotency gene expression, proliferation and differentiation ofneural stem cells. PNRP plays a role in in human tumors includingglioblastoma, breast cancer, prostate cancer and colorectal cancer (Yanget al., 2016b; Corsaro et al., 2016). In colorectal cancer PRNP has beenshown to contribute to epithelial-mesenchymal transition (Du et al.,2013). Over-expression of PNRP combined with MGr1-Ag/37LRP is predictiveof poor prognosis in gastric cancer (Zhou et al., 2014). PNRP expressionis related to redox state of cells and may participate in anti-oxidativedefense. Silencing PNRP has been shown to sensitize cancer cells toanticancer drugs in breast cancer and colon cancer (Sauer et al., 1999;Meslin et al., 2007; Park et al., 2015a; Yun et al., 2016).

PROM2 is specifically up-regulated in lung adenocarcinoma (Bao et al.,2016). PROM2 is expressed in elastofibromas and human prostate cancer.PROM2 is higher expressed in low aggressive prostate cancer and lowerexpressed in high aggressive prostate cancer (Yamazaki, 2007; Zhang etal., 2002). PROM2 is down-regulated in colon cancer (Deng et al., 2013).PROM2 expression may be used to differ between chromophobe renal cellcarcinoma and oncocytoma (Rohan et al., 2006).

RIPK4 was shown to be down-regulated in squamous cell carcinoma of theskin (Poligone et al., 2015). RIPK4 is associated with migration andinvasion in the tongue squamous cell carcinoma cell line Tca-8113,survival of diffuse large B-cell lymphoma and overall as well asdisease-free survival, progression and poor prognosis in cervicalsquamous cell carcinoma (Wang et al., 2014c; Liu et al., 2015; Kim etal., 2008b). RIPK4 is associated with familial pancreatic cancer (Lucitoet al., 2007). RIPK4 may be a potential diagnostic and independentprognostic biomarker for cervical squamous cell carcinoma and abiomarker for tongue cancer prognosis and treatment (Wang et al., 2014c;Liu et al., 2015).

RNASE7 expression is gradually decreased in cutaneous cancer (Scola etal., 2012). RNASE7 expression is affected by several leukemogenicprotein tyrosine kinases (Pierce et al., 2008).

RPL8 expression can be affected by MYC-induced nuclear antigen andnucleolar protein 66 (Chowdhury et al., 2014). RPL8 may be involved inosteosarcoma (Sun et al., 2015a; Yang and Zhang, 2013). RPL8 isregulated by NO-66 which is activated by MYC (Ge et al., 2012). Amutation in RPL8 is linked to Diamond-Blackfan anemia (Gazda et al.,2012). RPL8 expression may be linked to chemotherapy response (Salas etal., 2009). RPL8 is dysregulated in hepatocellular carcinoma (Liu etal., 2007). MHCII-dependent expression of RPL8 can be found in melanoma(Swoboda et al., 2007).

SERPINB5 is both a valuable molecular marker for the diagnosis and apredictor for the prognosis of many cancer types including breast, lung,head and neck, oral and prostate cancer (Marioni et al., 2009; Lonardoet al., 2010; Sager et al., 1996; Sheng, 2004). SERPINB5 acts as anendogenous regulator of HDAC1 activity and interacts with the p53 tumorsuppressor pathway (Maass et al., 2000; Kaplun et al., 2012).

SLC25A3 is de-regulated in chronic myeloid leukemia (Oehler et al.,2009). Depletion of SLC25A3 abolishes stress-induced mitochondrialtargeting of BAX (Buttner et al., 2011). SLC6A11 expression is reducedin rats with paclitaxel-induced neuropathic pain, a phenomenon which canbe observed in carcinoma patients treated with paclitaxel (Yadav et al.,2015). ALA and its methyl ester MAL are pro-drugs used in photodynamictherapy of skin cancer. Their uptake is mediated by SLC6A11 (Novak etal., 2011; Schulten et al., 2012; Baglo et al., 2013). Chronic treatmentof glioma cells with sodium valproate reduces SLC6A11 mRNA expression(Gao et al., 2003).

SLC6A15 is hyper-methylated and thereby down-regulated in colorectalcancer and may be a candidate biomarker for a stool-based assay (Kim etal., 2011; Mitchell et al., 2014).

SLC7A1 is constitutively expressed in acute myeloid leukemia blasts.These blasts show deficiencies in arginine-recycling pathway enzymesresulting in arginine accumulation and cell proliferation and survival(Mussai et al., 2015). SLC7A1 is over-expressed in colorectal cancerresulting in arginine accumulation and cell growth. Over-expression ofchromosome 13 genes is quite common in CRC (Camps et al., 2013; Lu etal., 2013). SLC7A1 may be used as a marker for macrophagedifferentiation. Its expression increases during the induction of THP1monocyte differentiation (Barilli et al., 2011). The growth of the MCF-7breast cancer cell line is dependent on L-arginine. It expresses SLC7A1and SLC7A1 knock-down results in reduced arginine uptake, decreased cellviability, and increased apoptosis (Abdelmagid et al., 2011). SLC7A1expression strongly correlates with the expression of Heme Oxygenase-1which is expressed in many cancers, promoting tumor growth and survival(Tauber et al., 2010). SLC7A1 is a direct target of the liver-specificmiR-122 which is down-regulated in hepatocellular carcinoma. miR-122down-regulation results in SLC7A1 up-regulation and increasedintracellular arginine levels. This pathway is also an importantmechanism for colorectal cancer-derived liver metastases (Kedde andAgami, 2008; Iino et al., 2013; Kishikawa et al., 2015). Activation ofprotein kinase C results in SLC7A1 internalization. Stress leads todifferential expression of SLC7A1 (Kakuda et al., 1998; Rotmann et al.,2006).

Loss of SUDS3 leads to altered cell morphology and increased cellmigration (Smith et al., 2012). SUDS3 is involved in thymocytedifferentiation (Lee et al., 2012). SUDS3 may have anti-tumor effects(Ramakrishna et al., 2012). USP17 de-ubiquitinates SUDS3 resulting inaltered SUDS3-associated HDAC activities in cancer (Ramakrishna et al.,2011). SUDS3 is involved in mitosis (Pondugula et al., 2009). SUDS3 isexpressed in breast cancer (Silveira et al., 2009). SUDS3 controlschromosome segregation and can interact with p53 (David et al., 2006).

TENM2 might be involved in age at menarche. Early AAM is associated withtype 2 diabetes mellitus, breast and ovarian cancer, and cardiovasculardiseases and late AAM is associated with low bone mineral density andpsychological disorders (Yermachenko and Dvornyk, 2016). There is aDOCK2-TENM2 gene fusion transcript in lung cancer patients living inhighly polluted regions (Yu et al., 2015a). TENM2 is expressed in themajority of malignant mesothelioma cells (Ziegler et al., 2012). TENM2may be down-regulated in esophageal squamous cell carcinoma (Kan et al.,2006).

Focal deletion of TGM5 in stage II colon cancer may be a driver for thisentity (Brosens et al., 2010). A TGM5 mutation occurring in non-smallcell lung cancer shows no difference between smokers and never-smokers(Yongjun Zhang et al., 2013; Broderick et al., 2009; Rafnar et al.,2011; Choi et al., 2016). Loss of TGFBR3 in prostate cancer alsodown-regulates TGM5 (Sharifi et al., 2007).

XIRP1 is mutated in the metastases of basal-like breast cancer (Hoadleyet al., 2016). XIRP1 promotor motif gene signature is enriched in triplenegative breast cancer compared to ER+ HER2− breast cancer (Willis etal., 2015). XIRP1 is up-regulated upon vitamin C treatment which alsodecreases cell growth in cancer (Marshall et al., 2012; Nagappan et al.,2013). XIRP1 is mutated in head and neck squamous cell carcinoma and maybe a tumor suppressor gene (Lee et al., 2010). XIRP1 is an oxidativestress associated gene (Baluchamy et al., 2010).

ZBED6 is a transcriptional repressor of IGF2 which is over-expressed incolorectal cancer and promotes cell proliferation. Knock-down of ZBED6affects the cell cycle and leads to enhanced cell growth in RKO cellline and reduced cell growth in HCT116 cells. ZBED6 is a transcriptionalrepressor of several genes involved in Wnt, Hippo, TGF-beta, EGFR, andPI3K signaling which are all involved in colorectal carcinogenesis(Markljung et al., 2009; Andersson, 2009; Andersson et al., 2010; Huanget al., 2014; Jiang et al., 2014; Clark et al., 2015; Akhtar et al.,2015).

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12, 13 or 14 or longer,and in case of MHC class II peptides (elongated variants of the peptidesof the invention) they can be as long as 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans, there are three different genetic loci that encode MHC classI molecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 5 Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA- DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy- Weinberg formula F = 1 −(1-Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles mightbe enriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype from Allele Populationallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that are A*02positive, whereas no selection for MHC class II allotypes is necessarydue to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and

(iiii) the alignment has to start at position 1 of the alignedsequences;

and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 91 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 91, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) Class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 91. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 91, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gin); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, lie, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) Class-I or—II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) Class-I or —II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acid whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptides accordingto SEQ ID NO: 4, 6, and 10 Position 1 2 3 4 5 6 7 8 9 10 SEQ ID NO. 4 1L D I N D N P P V Variants I L A M M I M L M A A A I A L A A V V I V L VA T T I T L T A Q Q I Q L Q A SEQ ID NO. 6 A L Y D A E L S Q M VariantsV I L A M V M I M L M A A V A I A L A A V V V I V L V A T V T I T L T AQ V Q I Q L Q A SEQ ID NO. 10 T L W P A T P P K A Variants V I L M V M IM L M A V A I A L A V V V I V L V T V T I T L T Q V Q I Q L Q

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations (extensions) of peptides of theinvention C-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2or 3 0 0 or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1or 2 C-terminus N-terminus 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof from 8 and 100, from 9 and 100, from 10 and 100, from 11 and 100,from 12 and 100, preferably from 8 and 30, and from 9 and 30, from 10and 30, from 11 and 30, from 12 and 30, most preferred from 8 and 14,from 9 and 14, from 10 and 14, from 11 and 14, from 12 and 14. Thepresent invention further provides peptides and variants of MHC class Iepitopes, wherein the peptide or variant has an overall length of namely8, 9, 10, 11, 12, 13, or 14 amino acids, in case of the elongated classII binding peptides the length can also be 15, 16, 17, 18, 19, 20, 21 or22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 91.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 91 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “li”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (www.sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID No: 1 to SEQ ID No: 156and has been synthetically produced (e.g. synthesized) as apharmaceutically acceptable salt. Methods to synthetically producepeptides are well known in the art. The salts of the peptides accordingto the present invention differ substantially from the peptides in theirstate(s) in vivo, as the peptides as generated in vivo are no salts. Thenon-natural salt form of the peptide mediates the solubility of thepeptide, in particular in the context of pharmaceutical compositionscomprising the peptides, e.g. the peptide vaccines as disclosed herein.A sufficient and at least substantial solubility of the peptide(s) isrequired in order to efficiently provide the peptides to the subject tobe treated.

Preferably, the salts are pharmaceutically acceptable salts of thepeptides. These salts according to the invention include alkaline andearth alkaline salts such as salts of the Hofmeister series comprisingas anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻, I⁻, SCN⁻ andas cations NH₄+, Rb+, K+, Na+, Cs+, Li+, Zn₂+, Mg₂+, Ca₂+, Mn₂+, Cu₂+and Ba₂+. Particularly salts are selected from (NH₄)₃PO₄, (NH₄)₂HPO₄,(NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃, NH₄ClO₄, NH₄I,NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO, Rb₄Cl, Rb₄Br,Rb₄NO₃, Rb₄ClO₄, Rb₄₁, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄, K₂SO₄, KCH₃COO,KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄, NaH₂PO₄, Na₂SO₄,NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂ Cs₃PO₄, Cs₂HPO₄,CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄, CsI, CsSCN,Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr, LiNO₃, LiClO₄,LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂, Mg₂SO₄, Mg(CH₃COO)₂,MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂, MnCl₂, Ca₃(PO₄),Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂, CaBr₂, Ca(NO₃)₂,Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂, BaSO₄,Ba(CH₃COO)₂, BaCI₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, and Ba(SCN)₂.Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄, KCl, NaCl,and CaCl₂, such as, for example, the chloride or acetate(trifluoroacetate) salts.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lukas et al.(Lukas et al., 1981) and by references as cited therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoracetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoracetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995) (cf. Example 1, FIGS. 1A-1Q).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from head and neck squamouscell carcinoma samples (N=17 A*02-positive samples) with thefragmentation patterns of corresponding synthetic reference peptides ofidentical sequences. Since the peptides were directly identified asligands of HLA molecules of primary tumors, these results provide directevidence for the natural processing and presentation of the identifiedpeptides on primary cancer tissue obtained from 14 head and necksquamous cell carcinoma patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from head and neck squamous cell carcinoma tissuesamples were purified and HLA-associated peptides were isolated andanalyzed by LC-MS (see examples). All TUMAPs contained in the presentapplication were identified with this approach on primary head and necksquamous cell carcinoma samples confirming their presentation on primaryhead and neck squamous cell carcinoma.

TUMAPs identified on multiple head and neck squamous cell carcinoma andnormal tissues were quantified using ion-counting of label-free LC-MSdata. The method assumes that LC-MS signal areas of a peptide correlatewith its abundance in the sample. All quantitative signals of a peptidein various LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

Besides over-presentation of the peptide, mRNA expression of theunderlying gene was tested. mRNA data were obtained via RNASeq analysesof normal tissues and cancer tissues (cf. Example 2, FIGS. 2A-2C). Anadditional source of normal tissue data was a database of publiclyavailable RNA expression data from around 3000 normal tissue samples(Lonsdale, 2013). Peptides which are derived from proteins whose codingmRNA is highly expressed in cancer tissue, but very low or absent invital normal tissues, were preferably included in the present invention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably head and neck squamous cell carcinoma thatover- or exclusively present the peptides of the invention. Thesepeptides were shown by mass spectrometry to be naturally presented byHLA molecules on primary human head and neck squamous cell carcinomasamples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy [insert normal tissue of major indication] cells or other normaltissue cells, demonstrating a high degree of tumor association of thesource genes (see Example 2). Moreover, the peptides themselves arestrongly over-presented on tumor tissue—“tumor tissue” in relation tothis invention shall mean a sample from a patient suffering from headand neck squamous cell carcinoma, but not on normal tissues (see Example1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. head and neck squamous cell carcinoma cellspresenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are inventive peptides capable of binding to TCRs andantibodies when presented by an MHC molecule. The present descriptionalso relates to nucleic acids, vectors and host cells for expressingTCRs and peptides of the present description; and methods of using thesame.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment, the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise, the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to an inventivepeptide-HLA molecule complex with a binding affinity (KD) of about 100μM or less, about 50 μM or less, about 25 μM or less, or about 10 μM orless. More preferred are high affinity TCRs having binding affinities ofabout 1 μM or less, about 100 nM or less, about 50 nM or less, about 25nM or less. Non-limiting examples of preferred binding affinity rangesfor TCRs of the present invention include about 1 nM to about 10 nM;about 10 nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM toabout 40 nM; about 40 nM to about 50 nM; about 50 nM to about 60 nM;about 60 nM to about 70 nM; about 70 nM to about 80 nM; about 80 nM toabout 90 nM; and about 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta heterodimeric TCRs of the present description may have a TRACconstant domain sequence and a TRBC1 or TRBC2 constant domain sequence,and the TRAC constant domain sequence and the TRBC1 or TRBC2 constantdomain sequence of the TCR may be linked by the native disulfide bondbetween Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a peptide-HLA molecule complex, which is at leastdouble that of a TCR comprising the unmutated TCR alpha chain and/orunmutated TCR beta chain. Affinity-enhancement of tumor-specific TCRs,and its exploitation, relies on the existence of a window for optimalTCR affinities. The existence of such a window is based on observationsthat TCRs specific for HLA-A2-restricted pathogens have KD values thatare generally about 10-fold lower when compared to TCRs specific forHLA-A2-restricted tumor-associated self-antigens. It is now known,although tumor antigens have the potential to be immunogenic, becausetumors arise from the individual's own cells only mutated proteins orproteins with altered translational processing will be seen as foreignby the immune system. Antigens that are upregulated or overexpressed (socalled self-antigens) will not necessarily induce a functional immuneresponse against the tumor: T-cells expressing TCRs that are highlyreactive to these antigens will have been negatively selected within thethymus in a process known as central tolerance, meaning that onlyT-cells with low-affinity TCRs for self-antigens remain. Therefore,affinity of TCRs or variants of the present description to peptides ofthe invention can be enhanced by methods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with peptide, incubating PBMCs obtained from the transgenic micewith tetramer-phycoerythrin (PE), and isolating the high avidity T-cellsby fluorescence activated cell sorting (FACS)-Calibur analysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “op-timal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3 (CD3 fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments, the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments, theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). In an aspect, a peptide described herein is in the form of apharmaceutically acceptable salt. In another aspect, a peptide in theform of a pharmaceutical salt is in crystalline form.

In an aspect, a pharmaceutically acceptable salt described herein refersto salts which possess toxicity profiles within a range that isacceptable for pharmaceutical applications.

As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an aspect, pharmaceutically acceptable salts may increase thesolubility and/or stability of peptides of described herein. In anotheraspect, pharmaceutical salts described herein may be prepared byconventional means from the corresponding carrier peptide or complex byreacting, for example, the appropriate acid or base with peptides orcomplexes as described herein. In another aspect, the pharmaceuticallyacceptable salts are in crystalline form or semi-crystalline form. Inyet another aspect, pharmaceutically acceptable salts may include, forexample, those described in Handbook of Pharmaceutical Salts:Properties, Selection, and Use By P. H. Stahl and C. G. Wermuth(Wiley-VCH 2002) and L. D. Bighley, S. M. Berge, D. C. Monkhouse, in“Encyclopedia of Pharmaceutical Technology”. Eds. J. Swarbrick and J. C.Boylan, Vol. 13, Marcel Dekker, Inc., New York, Basel, Hong Kong 1995,pp. 453-499, each of these references is herein incorporated byreference in their entirety.

The pharmaceutically acceptable salt form of the peptides of the presentinvention increases stability over native (naked) peptides, i.e., not inthe form of a pharmaceutically acceptable salt or otherwise modified. Asused herein, increased stability includes measurable decrease orreduction of the following: deamidation, oxidation, hydrolysis,disulfide interchange, racemization and hydrolysis. The stability can beevaluated on a peptide as a finished product or during the productionprocedure or during storage. Preferably the peptide in the form of apharmaceutically acceptable salt has sufficient stability in vitro toallow storage at a commercially relevant temperature, such as betweenabout 0° C. and about 60° C., for a commercially relevant period oftime, such as at least one week, preferably at least one month, morepreferably at least three months, and most preferably at least sixmonths. Stability can be measured using any physiochemicalcharacterization techniques known to those skilled in the art, such as,for example high pressure liquid chromatography (HPLC).

Further, stability of the peptide salts can be determined by lessenzymatic degradation (such as by aminopeptidases, exopeptidases, andsynthetases) in plasma, and/or having an improved in vivo half life,compared to native peptide. It has been determined that theintracellular half-life of native 9-mer peptides generated for MHC Iantigen presentation is only a few seconds, unless they are bound byother molecules such as chaperons or MHC I molecules. Reits et al.,Immunity. 2004; 20: 495-506.

In addition, the pharmaceutically acceptable salts of the peptides ofthe present invention exhibit improved solubility and stability inaqueous solutions. Berge et al., Pharmaceutical Salts, J. Pharm. Sci.1977; 66(1): 1-19. Preferably the peptide in the form of apharmaceutically acceptable salt in an aqueous solution is stable for atleast 4 days, at least 5 days, at least 6 days, and more preferably atleast 7 days.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 91, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbas and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment, the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also, cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,Sunitinib, Bevacizumab®, Celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, Sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, Sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

In an aspect, peptides or other molecules described herein may becombined with an aqueous carrier. In an aspect, the aqueous carrier isselected from ion exchangers, alumina, aluminum stearate, magnesiumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,dicalcium phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyvinylpyrrolidone-vinyl acetate, cellulose-based substances (e.g.,microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropyl methylcellulosePhthalate), starch, lactose monohydrate, mannitol, trehalose sodiumlauryl sulfate, and crosscarmellose sodium, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, polymethacrylate, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

In an aspect, the aqueous carrier contains multiple components, such aswater together with a non-water carrier component, such as thosecomponents described herein. In another aspect, the aqueous carrier iscapable of imparting improved properties when combined with a peptide orother molecule described herein, for example, improved solubility,efficacy, and/or improved immunotherapy. In addition, the compositioncan contain excipients, such as buffers, binding agents, blastingagents, diluents, flavors, lubricants, etc. A “pharmaceuticallyacceptable diluent,” for example, may include solvents, bulking agents,stabilizing agents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likewhich are physiologically compatible. Examples of pharmaceuticallyacceptable diluents include one or more of saline, phosphate bufferedsaline, dextrose, glycerol, ethanol, and the like as well ascombinations thereof. In many cases it will be preferable to include oneor more isotonic agents, for example, sugars such as trehalose andsucrose, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Pharmaceutically acceptable substances such as wettingor minor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, are also within the scope of thepresent invention. In addition, the composition can contain excipients,such as buffers, binding agents, blasting agents, diluents, flavors, andlubricants.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore, differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment, a scaffold is ableto activate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting another peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example, afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed, aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 91,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 91, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 91 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:91 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 91, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) Class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 91.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (li), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of head and neck squamous cellcarcinoma.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 91 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are head and neck squamous cellcarcinoma cells or other solid or hematological tumor cells such asacute myelogenous leukemia, breast cancer, bile duct cancer, braincancer, chronic lymphocytic leukemia, colorectal carcinoma, esophagealcancer, gallbladder cancer, gastric cancer, hepatocellular cancer,melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovariancancer, pancreatic cancer, prostate cancer, renal cell cancer, smallcell lung cancer, urinary bladder cancer and uterine cancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of head and neck squamous cell carcinoma. The presentinvention also relates to the use of these novel targets for cancertreatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a head and neck squamous cellcarcinoma marker (poly)peptide, delivery of a toxin to a head and necksquamous cell carcinoma cell expressing a cancer marker gene at anincreased level, and/or inhibiting the activity of a head and necksquamous cell carcinoma marker polypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length head and neck squamous cell carcinoma markerpolypeptides or fragments thereof may be used to generate the antibodiesof the invention. A polypeptide to be used for generating an antibody ofthe invention may be partially or fully purified from a natural source,or may be produced using recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 91polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the head and neck squamous cellcarcinoma marker polypeptide used to generate the antibody according tothe invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating head and necksquamous cell carcinoma, the efficacy of the therapeutic antibody can beassessed in various ways well known to the skilled practitioner. Forinstance, the size, number, and/or distribution of cancer in a subjectreceiving treatment may be monitored using standard tumor imagingtechniques. A therapeutically-administered antibody that arrests tumorgrowth, results in tumor shrinkage, and/or prevents the development ofnew tumors, compared to the disease course that would occur in theabsence of antibody administration, is an efficacious antibody fortreatment of cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 91, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptide complexes to the surface of polystyrene particles (microbeads)by biotin: streptavidin biochemistry. This system permits the exactcontrol of the MHC density on aAPCs, which allows to selectively elicithigh- or low-avidity antigen-specific T cell responses with highefficiency from blood samples. Apart from MHC: peptide complexes, aAPCsshould carry other proteins with co-stimulatory activity like anti-CD28antibodies coupled to their surface. Furthermore, such aAPC-basedsystems often require the addition of appropriate soluble factors, e. g.cytokines, like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition, plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 91.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor, it is expressed. By “over-expressed” the inventorsmean that the polypeptide is present at a level at least 1.2-fold ofthat present in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore, anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from head and necksquamous cell carcinoma, the medicament of the invention is preferablyused to treat head and neck squamous cell carcinoma.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue of head andneck squamous cell carcinoma patients with various HLA-A HLA-B and HLA-Calleles. It may contain MHC class I and MHC class II peptides orelongated MHC class I peptides. In addition to the tumor associatedpeptides collected from several head and neck squamous cell carcinomatissues, the warehouse may contain HLA-A*02 and HLA-A*24 markerpeptides. These peptides allow comparison of the magnitude of T-cellimmunity induced by TUMAPS in a quantitative manner and hence allowimportant conclusion to be drawn on the capacity of the vaccine toelicit anti-tumor responses. Secondly, they function as importantpositive control peptides derived from a “non-self” antigen in the casethat any vaccine-induced T-cell responses to TUMAPs derived from “self”antigens in a patient are not observed. And thirdly, it may allowconclusions to be drawn, regarding the status of immunocompetence of thepatient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, head and neck squamous cellcarcinoma samples from patients and blood from healthy donors wereanalyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (head andneck squamous cell carcinoma) compared with a range of normal organs andtissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.

4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs

5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.

6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromhead and neck squamous cell carcinoma patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory, an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients' tumor and,where possible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andover presentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from head and neck squamous cell carcinoma cells andsince it was determined that these peptides are not or at lower levelspresent in normal tissues, these peptides can be used to diagnose thepresence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for head and neck squamous cell carcinoma. Presence of groupsof peptides can enable classification or sub-classification of diseasedtissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

FIGS. 1A through 1Q show the over-presentation of various peptides innormal tissues (white bars) and head and neck squamous cell carcinoma(black bars). FIG. 1A) Gene symbols: KRT6C, KRT6A, KRT6B, Peptide:GLAGGFGGPGFPV (SEQ ID NO.: 1); Tissues from left to right: 6 adiposetissues, 8 adrenal glands, 1 bile duct, 24 blood cells, 15 bloodvessels, 10 bone marrows, 15 brains, 7 breasts, 11 esophagi, 2 eyes, 6gallbladders, 16 hearts, 17 kidneys, 27 large intestines, 24 livers, 49lungs, 7 lymph nodes, 12 nerves, 5 ovaries, 15 pancreases, 6 parathyroidglands, 3 peritoneums, 7 pituitary glands, 10 placentas, 3 pleuras, 11prostates, 9 skeletal muscles, 11 skins, 16 small intestines, 13spleens, 9 stomachs, 8 testes, 3 thymi, 8 thyroid glands, 18 tracheas, 7ureters, 8 urinary bladders, 6 uteri, 12 head-and-necks, 17 HNSCC. FIG.1B) Gene symbols: KRT6C, KRT6A, KRT6B, Peptide: SLYGLGGSKRISI (SEQ IDNO.: 3); Tissues from left to right: 6 adipose tissues, 8 adrenalglands, 1 bile duct, 24 blood cells, 15 blood vessels, 10 bone marrows,15 brains, 7 breasts, 11 esophagi, 2 eyes, 6 gallbladders, 16 hearts, 17kidneys, 27 large intestines, 24 livers, 49 lungs, 7 lymph nodes, 12nerves, 5 ovaries, 15 pancreases, 6 parathyroid glands, 3 peritoneums, 7pituitary glands, 10 placentas, 3 pleuras, 11 prostates, 9 skeletalmuscles, 11 skins, 16 small intestines, 13 spleens, 9 stomachs, 8testes, 3 thymi, 8 thyroid glands, 18 tracheas, 7 ureters, 8 urinarybladders, 6 uteri, 12 head-and-necks, 17 HNSCC. FIG. 1C) Gene symbol:KRT5, Peptide: STASAITPSV (SEQ ID NO.: 9); Tissues from left to right: 6adipose tissues, 8 adrenal glands, 1 bile duct, 24 blood cells, 15 bloodvessels, 10 bone marrows, 15 brains, 7 breasts, 11 esophagi, 2 eyes, 6gallbladders, 16 hearts, 17 kidneys, 27 large intestines, 24 livers, 49lungs, 7 lymph nodes, 12 nerves, 5 ovaries, 15 pancreases, 6 parathyroidglands, 3 peritoneums, 7 pituitary glands, 10 placentas, 3 pleuras, 11prostates, 9 skeletal muscles, 11 skins, 16 small intestines, 13spleens, 9 stomachs, 8 testes, 3 thymi, 8 thyroid glands, 18 tracheas, 7ureters, 8 urinary bladders, 6 uteri, 12 head-and-necks, 17 HNSCC. FIG.1D) Gene symbol: SLC25A3, Peptide: FVAGYIAGV (SEQ ID NO.: 61); Tissuesfrom left to right: 3 cell lines (2 kidney, 1 pancreas), 7 normaltissues (1 adrenal gland, 1 colon, 2 lymph nodes, 1 placenta, 2spleens), 36 cancer tissues (5 leukocytic leukemia cancers, 3 braincancers, 2 breast cancers, 1 esophageal cancer, 1 gallbladder cancer, 5head-and-neck cancers, 1 kidney cancer, 1 liver cancer, 8 lung cancers,4 lymph node cancers, 3 ovarian cancers, 2 stomach cancers). FIGS. 1E to1Q show the over-presentation of various peptides in different cancertissues (black dots). Upper part: Median MS signal intensities fromtechnical replicate measurements are plotted as dots for single HLA-A*02positive normal (grey dots) and tumor samples (black dots) on which thepeptide was detected. Tumor and normal samples are grouped according toorgan of origin, and box-and-whisker plots represent median, 25th and75th percentile (box), and minimum and maximum (whiskers) of normalizedsignal intensities over multiple samples. Normal organs are orderedaccording to risk categories (blood cells, blood vessels, brain, liver,lung: high risk, grey dots; reproductive organs, breast, prostate: lowrisk, grey dots; all other organs: medium risk; grey dots). Lower part:The relative peptide detection frequency in every organ is shown asspine plot. Numbers below the panel indicate number of samples on whichthe peptide was detected out of the total number of samples analyzed foreach organ (N=526 for normal samples, N=562 for tumor samples). If thepeptide has been detected on a sample but could not be quantified fortechnical reasons, the sample is included in this representation ofdetection frequency, but no dot is shown in the upper part of thefigure. Tissues (from left to right): Normal samples: blood cells;bloodvess (blood vessels); brain; heart; liver; lung; adipose (adiposetissue); adren.gl. (adrenal gland); bile duct; bladder; BM (bonemarrow); cartilage; esoph (esophagus); eye; gallb (gallbladder);head&neck; kidney; large_int (large intestine); LN (lymph node); nerve;pancreas; parathyr (parathyroid gland); perit (peritoneum); pituit(pituitary); pleura; skel.mus (skeletal muscle); skin; small_int (smallintestine); spleen; stomach; thyroid; trachea; ureter; breast; ovary;placenta; prostate; testis; thymus; uterus. Tumor samples: AML: acutemyeloid leukemia; BRCA: breast cancer; CCC: cholangiocellular carcinoma;CLL: chronic lymphocytic leukemia; CRC: colorectal cancer; GBC:gallbladder cancer; GBM: glioblastoma; GC: gastric cancer; GEJC: stomachcardia esophagus, cancer; HCC: hepatocellular carcinoma; HNSCC:head-and-neck cancer; MEL: melanoma; NHL: non-hodgkin lymphoma; NSCLC:non-small cell lung cancer; OC: ovarian cancer; OSCAR: esophagealcancer; PACA: pancreatic cancer; PRCA: prostate cancer; RCC: renal cellcarcinoma; SCLC: small cell lung cancer; UBC: urinary bladder carcinoma;UEC: uterine and endometrial cancer. FIG. 1E) Gene symbols: KRT6C,KRT6A, KRT1, KRT6B, KRT75, KRT5, Peptide: PVCPPGGIQEV (SEQ ID NO.: 2),FIG. 1F) Gene symbol: PKP1, Peptide: SMLNNIINL (SEQ ID NO.: 15), FIG.1G) Gene symbol: PRKDC, Peptide: GLIEWLENTV (SEQ ID NO.: 45), FIG. 1H)Gene symbol: ATP5G2, ATP5G1, ATP5G3, Peptide: AILGFALSEA (SEQ ID NO.:57), FIG. 1I) Gene symbol: ITGB4, Peptide: SLSDIQPCL (SEQ ID NO.: 58),FIG. 1J) Gene symbol: KRT5, Peptide: ALMDEINFMKM (SEQ ID NO.: 63), FIG.1K) Gene symbol: ESRP2, Peptide: ALASAPTSV (SEQ ID NO.: 75), FIG. 1L)Gene symbol: PARP9, Peptide: ILFDEVLTFA (SEQ ID NO.: 76), FIG. 1M) Genesymbol: MCM4, Peptide: QLLQYVYNL (SEQ ID NO.: 83), FIG. 1N) Gene symbol:FHAD1, Peptide: QLIEKITQV (SEQ ID NO.: 85), FIG. 1O) Gene symbol: PLEC,Peptide: ALPEPSPAA (SEQ ID NO.: 87), FIG. 1P) Gene symbol: G3BP1,Peptide: TLNDGVVVQV (SEQ ID NO.: 90), FIG. 1Q) Gene symbol: ODC1,Peptide: MLFENMGAYTV (SEQ ID NO.: 91).

FIGS. 2A through 2C show exemplary expression profiles of source genesof the present invention that are highly over-expressed or exclusivelyexpressed in head and neck squamous cell carcinoma in a panel of normaltissues (white bars) and 15 head and neck squamous cell carcinomasamples (black bars). FIG. 2A) Gene symbol: PGLYRP4, Peptide: AIYEGVGWNV(SEQ ID NO.: 33); FIG. 2B) Gene symbol: PAPL, Peptide: KLLPGVQYV (SEQ IDNO.: 38); FIG. 2C) Gene symbols: LGALS7, LGALS7B, Peptide: RLVEVGGDVQL(SEQ ID NO.: 53).

FIG. 3 shows exemplary immunogenicity data: flow cytometry results afterpeptide-specific multimer staining.

FIG. 4 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*02+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*02 in complex withSeq ID NO.: 17 peptide (A, left panel), Seq ID NO.: 28 peptide (B, leftpanel) and Seq ID NO.: 29 peptide (C, left panel), respectively. Afterthree cycles of stimulation, the detection of peptide-reactive cells wasperformed by 2D multimer staining with A*02/Seq ID NO.: 17 (A), A*02/SeqID NO.: 28 (B) or A*02/Seq ID NO.: 29 (C). Right panels (A, B and C)show control staining of cells stimulated with irrelevant A*02/peptidecomplexes. Viable singlet cells were gated for CD8+ lymphocytes. Booleangates helped excluding false-positive events detected with multimersspecific for different peptides. Frequencies of specific multimer+ cellsamong CD8+ lymphocytes are indicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained from: Asterand (Detroit, Mich.,USA & Royston, Herts, UK); ProteoGenex Inc. (Culver City, Calif., USA).Normal tissues were obtained from Asterand (Detroit, Mich., USA &Royston, Herts, UK); Bio-Options Inc. (Brea, Calif., USA); BioServe(Beltsville, Md., USA); Capital BioScience Inc. (Rockville, Md., USA);Geneticist Inc. (Glendale, Calif., USA); Kyoto Prefectural University ofMedicine (KPUM) (Kyoto, Japan); ProteoGenex Inc. (Culver City, Calif.,USA); Tissue Solutions Ltd (Glasgow, UK); University Hospital Geneva(Geneva, Switzerland); University Hospital Heidelberg (Heidelberg,Germany); University Hospital Tubingen (Tubingen, Germany). Writteninformed consents of all patients had been given before surgery orautopsy. Tissues were shock-frozen immediately after excision and storeduntil isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, —B, C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7m C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOP5 strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the Orbitrap(R=30 000), which was followed by MS/MS scans also in the Orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus, each identifiedpeptide can be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide, a presentation profile was calculatedshowing the mean sample presentation as well as replicate variations.The profiles juxtapose head and neck squamous cell carcinoma samples toa baseline of normal tissue samples. Presentation profiles of exemplaryover-presented peptides are shown in FIGS. 1A-1Q. Presentation scoresfor exemplary peptides are shown in Table 8.

TABLE 8 Presentation scores. The table lists peptidesthat are very highly over-presented on tumorscompared to a panel of normal tissues (+++),highly over-presented on tumors compared to apanel of normal tissues (++) or over-presentedon tumors compared to a panel of normal tissues(+). The panel of normal tissues consideredrelevant for comparison with tumors consistedof: adipose tissue, adrenal gland, bile duct,blood cells, blood vessel, bone marrow, brain,esophagus, eye, gallbladder, head-and-neck,heart, kidney, large intestine, liver, lung,lymph node, nerve, pancreas, parathyroid gland,peritoneum, pituitary, pleura, skeletal muscle,skin, small intestine, spleen, stomach, thymus,thyroid gland, trachea, ureter, urinary bladder. SEQ ID No SequencePeptide Presentation  1 GLAGGFGGPGFPV +++  2 PVCPPGGIQEV +++  3SLYGLGGSKRISI +++  4 ILDINDNPPV +++  5 VCPPGGIQEV +++  6 ALYDAELSQM +++ 7 ALEEANADLEV +++  8 AQLNIGNVLPV +++  9 STASAITPSV +++ 10 TLWPATPPKA+++ 11 VLFSSPPVI +++ 12 TLTDEINFL +++ 13 SLVSYLDKV +++ 14 RIMEGIPTV +++15 SMLNNIINL + 16 ALKDSVQRA +++ 17 SIWPALTQV +++ 19 ALAKLLPLL +++ 20YLINEIDRIRA +++ 21 FLHEPFSSV +++ 22 KLPEPCPSTV +++ 23 SLPESGLLSV +++ 24LLIAINPQV +++ 25 SLCPPGGIQEV +++ 26 TLVDENQSWYL +++ 27 YLAEPQWAV +++ 28AVDPVSGSLYV +++ 29 RLLPDLDEV +++ 30 TLASLGYAVV +++ 31 HLATVKLLV +++ 32IQDAEGAIHEV +++ 33 AIYEGVGWNV +++ 34 ALDTFSVQV +++ 35 ALVGDVILTV +++ 36GLWSSIFSL +++ 37 ILLEDVFQL +++ 38 KLLPGVQYV +++ 39 LLPEDDTRDNV +++ 40LLTPLNLQI +++ 41 RLNGEGVGQVNISV +++ 42 ALYTSGHLL +++ 43 AVLGGKLYV +++ 44GLGDDSFPI +++ 45 GLIEWLENTV +++ 46 GLISSIEAQL +++ 47 QLLEGELETL +++ 48YLLDYPNNL +++ 49 YLWEAHTNI +++ 50 ALSNVVHKV +++ 51 FLIPSIIFA +++ 52LLFTGLVSGV +++ 53 RLVEVGGDVQL +++ 54 RLSGEGVGPV +++ 55 VLNVGVAEV +++ 56FLQLETEQV +++ 58 SLSDIQPCL +++ 60 SLGNFKDDLL +++ 61 FVAGYIAGV +++ 62ILSSACYTV +++ 63 ALMDEINFMKM ++ 67 AQLNLIWQL + 69 YVMESMTYL + 70FLFPAFLTA +++ 71 SLFPYVVLI +++ 72 SLDGNPLAV +++ 73 YIDPYKLLPL +++ 74SLTSFLISL +++ 75 ALASAPTSV +++ 78 VLYGDVEEL +++ 79 GLHQDFPSVVL + 81VLAENPDIFAV +++ 82 VLDINDNPPV +++ 83 QLLQYVYNL +++ 84 ALMAGCIQEA +++ 85QLIEKITQV +++ 86 SLQERQVFL +++ 88 LMAPAPSTV ++ 90 TLNDGVVVQV + 91MLFENMGAYTV +

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH(Heidelberg, Germany); Bio-Options Inc. (Brea, Calif., USA); BioServe(Beltsville, Md., USA); Capital BioScience Inc. (Rockville, Md., USA);Geneticist Inc. (Glendale, Calif., USA); Istituto Nazionale Tumori“Pascale” (Naples, Italy); ProteoGenex Inc. (Culver City, Calif., USA);University Hospital Heidelberg (Heidelberg, Germany). Total RNA fromtumor tissues for RNASeq experiments was obtained from: Asterand(Detroit, Mich., USA & Royston, Herts, UK); ProteoGenex Inc. (CulverCity, Calif., USA).

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNASeq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNASeq) by CeGaT (TObingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (IlluminaInc., San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolar and sequenced on the Illumina HiSeq2500 sequencer according to the manufacturer's instructions, generating50 bp single end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of the ensemblsequence database (Ensembl77). Exon reads are normalized for exon lengthand alignment size to obtain RPKM values.

Exemplary expression profiles of source genes of the present inventionthat are highly over-expressed or exclusively expressed in head and necksquamous cell carcinoma are shown in FIGS. 2A-2C. Expression scores forfurther exemplary genes are shown in Table 9.

TABLE 9 Expression scores. The table lists peptidesfrom genes that are very highly over-expressedin tumors compared to a panel of normaltissues (+++), highly over-expressed in tumorscompared to a panel of normal tissues (++) orover-expressed in tumors compared to a panel ofnormal tissues (+). The baseline for this scorewas calculated from measurements of thefollowing relevant normal tissues: adiposetissue, adrenal gland, artery, bile duct, bloodcells, bone marrow, brain, cartilage, colon,esophagus, eye, gallbladder, head-and-neck andsalivary gland, heart, kidney, liver, lung,lymph node, pancreas, parathyroid gland,peripheral nerve, peritoneum, pituitary, pleura,rectum, skeletal muscle, skin, small intestine,spleen, stomach, thyroid gland, trachea, ureter,urinary bladder, and vein. In case expressiondata for several samples of the same tissue typewere available, the arithmetic mean of allrespective samples was used for the calculation. SEQ Gene ID No SequenceExpression  1 GLAGGFGGPGFPV +++  2 PVCPPGGIQEV +++  3 SLYGLGGSKRISI +++ 4 ILDINDNPPV +++  5 VCPPGGIQEV +++  6 ALYDAELSQM +++  7 ALEEANADLEV +++ 9 STASAITPSV +++ 12 TLTDEINFL +++ 14 RIMEGIPTV +++ 15 SMLNNIINL +++ 19ALAKLLPLL +++ 20 YLINEIDRIRA ++ 21 FLHEPFSSV +++ 24 LLIAINPQV +++ 26TLVDENQSWYL +++ 27 YLAEPQWAV + 33 AIYEGVGWNV +++ 34 ALDTFSVQV ++ 35ALVGDVILTV + 36 GLWSSIFSL +++ 38 KLLPGVQYV +++ 39 LLPEDDTRDNV +++ 40LLTPLNLQI +++ 41 RLNGEGVGQVNISV +++ 49 YLWEAHTNI + 51 FLIPSIIFA +++ 53RLVEVGGDVQL +++ 54 RLSGEGVGPV +++ 59 YLQNEVFGL +++ 60 SLGNFKDDLL +++ 62ILSSACYTV +++ 63 ALMDEINFMKM +++ 82 VLDINDNPPV +++ 91 MLFENMGAYTV ++

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*0201 restricted TUMAPs ofthe invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotech, Bergisch-Gladbach, Germany) of healthy donorsobtained from the University clinics Mannheim, Germany, after informedconsent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nirnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 157) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.158), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Head and Neck Squamous Cell CarcinomaPeptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 2peptides of the invention are shown in FIG. 3 together withcorresponding negative controls. Additional exemplary flow cytometryresults after TUMAP-specific multimer staining for 3 peptides of theinvention are shown in FIG. 4 together with corresponding negativecontrols. Results for 17 peptides from the invention are summarized inTable 10A. Additional results for 17 peptides from the invention aresummarized in Table 10B.

TABLE 10A in vitro immunogenicity of HLA class I peptidesof the invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention. <20% = +; 20%-49% =++; 50%-69% = +++; >= 70% = ++++ Seq ID No Peptide Code Sequence WellsDonors  98 RAD54B-001 SLYKGLLSV ++ ++++ 101 C4orf36-001 GLLPSAESIKL +++++ 105 KRT-010 STYGGGLSV + ++++ 108 KRT5-001 SLYNLGGSKRISI + ++++ 109IGF2BP3-001 KIQEILTQV + +++ 113 PKP1-002 NLMASQPQL +++ ++++ 114 TP6-001VLVPYEPPQV + ++++ 118 KRT-006 TLLQEQGTKTV + ++ 123 GJB5-001 LLSGDLIFL ++++++ 127 FHL2-001 SLFGKKYIL ++ ++++ 135 DNMT3B-001 GLFSQHFNL + ++ 137LOC-002 GLAPFLLNAV + ++++ 143 FAP-003 YVYQNNIYL + ++ 145 TMEM222-001LLYGKYVSV ++ ++++ 147 DNMT1-001 ILMDPSPEYA +++ ++++ 150 NLRP2-001ILAEEPIYIRV +++ ++++ 154 BDH1-001 KMWEELPEVV + ++++

TABLE 10B In vitro immunogenicity of HLA class I peptidesof the invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant forHLA-A*02 restricted peptides of the invention.Results of in vitro immunogenicity experimentsare indicated. Percentage of positive wellsand donors (among evaluable) are summarizedas indicated <20% = +; 20%-49% = ++; 50%-69% = +++; >+ 70% = ++++ SEQ ID No Sequence Wells positive [%]  1 GLAGGFGGPGFPV +  3SLYGLGGSKRISI +  8 AQLNIGNVLPV + 10 TLWPATPPKA + 11 VLFSSPPVI ++ 12TLTDEINFL + 13 SLVSYLDKV ++ 16 ALKDSVQRA + 17 SIWPALTQV ++++ 18YLYPDLSRL + 19 ALAKLLPLL ++ 20 YLINEIDRIRA + 26 TLVDENQSWYL ++ 28AVDPVSGSLYV +++ 29 RLLPDLDEV ++ 30 TLASLGYAVV +++ 82 VLDINDNPPV +

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizes (trifluoro acetate salt) in purities of >50%. All TUMAPs arepreferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 3700 in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100-fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores. Binding of HLA-classI restricted peptides to HLA-A*02:01 was rangedby peptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++SEQ ID No Sequence Peptide exchange  1 GLAGGFGGPGFPV ++++  2 PVCPPGGIQEV+++  3 SLYGLGGSKRISI +++  4 ILDINDNPPV +++  5 VCPPGGIQEV +++  6 ALYDAELSQM +++  7 ALEEANADLEV +++  8 AQLNIGNVLPV ++++  9 STASAITPSV ++10 TLWPATPPKA +++ 11 VLFSSPPVI ++++ 12 TLTDEINFL ++++ 13 SLVSYLDKV ++++14 RIMEGIPTV ++++ 15 SMLNNIINL +++ 16 ALKDSVQRA +++ 17 SIWPALTQV ++++ 18YLYPDLSRL ++++ 19 ALAKLLPLL ++++ 20 YLINEIDRIRA ++++ 21 FLHEPFSSV ++++22 KLPEPCPSTV +++ 23 SLPESGLLSV +++ 24 LLIAINPQV ++++ 25 SLCPPGGIQEV++++ 26 TLVDENQSWYL +++ 27 YLAEPQWAV ++++ 28 AVDPVSGSLYV ++++ 29RLLPDLDEV ++++ 30 TLASLGYAVV +++ 31 HLATVKLLV ++++ 32 IQDAEGAIHEV ++++33 AIYEGVGWNV ++++ 34 ALDTFSVQV ++++ 35 ALVGDVILTV ++++ 36 GLWSSIFSL++++ 37 ILLEDVFQL ++++ 38 KLLPGVQYV ++++ 39 LLPEDDTRDNV +++ 40 LLTPLNLQI+++ 41 RLNGEGVGQVNISV ++ 42 ALYTSGHLL ++++ 43 AVLGGKLYV ++++ 44GLGDDSFPI ++++ 45 GLIEWLENTV ++++ 46 GLISSIEAQL ++++ 47 QLLEGELETL +++48 YLLDYPNNL +++ 49 YLWEAHTNI ++++ 50 ALSNVVHKV ++++ 51 FLIPSIIFA ++++52 LLFTGLVSGV +++ 53 RLVEVGGDVQL ++++ 54 RLSGEGVGPV +++ 55 VLNVGVAEV +++56 FLQLETEQV ++++ 57 AILGFALSEA ++++ 58 SLSDIQPCL +++ 59 YLQNEVFGL +++60 SLGNFKDDLL +++ 61 FVAGYIAGV ++++ 62 ILSSACYTV ++++ 63 ALMDEINFMKM++++ 64 KILEJLFVJL +++ 65 ALWGFFPVLL ++++ 66 TLLSEIAEL ++++ 67 AQLNLIWQL++++ 68 KILEMDDPRA ++ 69 YVMESMTYL ++++ 70 FLFPAFLTA ++++ 71 SLFPYVVLI++++ 72 SLDGNPLAV ++++ 73 YIDPYKLLPL +++ 74 SLTSFLISL +++ 75 ALASAPTSV++++ 76 ILFDEVLTFA ++++ 77 SLRAFLMPI ++ 78 VLYGDVEEL +++ 79 GLHQDFPSVVL+++ 80 GLYGIKDDVFL ++++ 81 VLAENPDIFAV +++ 82 VLDINDNPPV +++ 83QLLQYVYNL ++++ 84 ALMAGCIQEA ++++ 85 QLIEKITQV +++ 86 SLQERQVFL +++ 87ALPEPSPAA +++ 88 LMAPAPSTV +++ 89 VLDEGLTSV ++++ 90 TLNDGVVVQV ++++ 91MLFENMGAYTV ++++

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1. A method of treating cancer in a HLA-A*02+ patient, wherein saidcancer comprises cancer cells that overexpress AHCTF1 and present attheir surface in complex with an MHC class I molecule a peptideconsisting of the amino acid sequence VLDEGLTSV (SEQ ID NO: 89), saidmethod comprising administering to said patient an effective amount ofactivated antigen-specific CD8+ cytotoxic T cells to kill the cancercells, wherein said activated antigen-specific CD8+ cytotoxic T cellsare produced by contacting in vitro CD8+ cytotoxic T cells with anantigen presenting cell presenting at its surface in complex with an MHCclass I molecule a peptide consisting of the amino acid sequenceVLDEGLTSV (SEQ ID NO: 89), wherein said cancer is selected from smallcell lung cancer, renal cell carcinoma, chronic lymphatic leukemia,non-Hodgkin lymphoma, breast cancer, melanoma, urinary bladder cancer,uterine cancer, and gastric cancer.
 2. The method of claim 1, whereinthe cytotoxic T cells produced by contacting CD8+ cytotoxic T cells withsaid antigen presenting cell are cytotoxic T cells autologous to thepatient.
 3. The method of claim 1, wherein the cytotoxic T cellsproduced by contacting CD8+ cytotoxic T cells with said antigenpresenting cell are cytotoxic T cells obtained from a healthy donor. 4.The method of claim 1, wherein the cytotoxic T cells produced bycontacting CD8+ cytotoxic T cells with said antigen presenting cell arecytotoxic T cells isolated from tumor infiltrating lymphocytes orperipheral blood mononuclear cells.
 5. The method of claim 1, whereinthe cytotoxic T cells produced by contacting CD8+ cytotoxic T cells withsaid antigen presenting cell are expanded in vitro before beingadministered to the patient.
 6. The method of claim 5, wherein thecytotoxic T cells are expanded in vitro in the presence of an anti-CD28antibody and IL-12.
 7. The method of claim 1, wherein the effectiveamount of activated antigen-specific CD8+ cytotoxic T1 cells to kill thecancer cells are administered in the form of a composition.
 8. Themethod of claim 7, wherein said composition further comprises anadjuvant.
 9. The method of claim 8, wherein said adjuvant is selectedfrom agonistic anti-CD40 antibody, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, interferon-alpha, interferon-beta, CpGoligonucleotides, poly-(I:C), RNA, sildenafil, particulate formulationswith poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1,IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
 10. The methodof claim 8, wherein the adjuvant comprises IL-2.
 11. The method of claim8, wherein the adjuvant comprises IL-21.
 12. The method of claim 1,wherein the cancer is small cell lung cancer.
 13. The method of claim 1,wherein the cancer is renal cell carcinoma.
 14. The method of claim 1,wherein the cancer is chronic lymphatic leukemia.
 15. The method ofclaim 1, wherein the cancer is non-Hodgkin lymphoma.
 16. The method ofclaim 1, wherein the cancer is breast cancer.
 17. The method of claim 1,wherein the cancer is melanoma.
 18. The method of claim 1, wherein thecancer is urinary bladder cancer.
 19. The method of claim 1, wherein thecancer is uterine cancer.
 20. The method of claim 1, wherein the canceris gastric cancer.